Compositions and Method for Inhibiting Hepcidin Antimicrobial Peptide (HAMP) or HAMP-Related Gene Expression

ABSTRACT

The invention relates to lipid formulated double-stranded ribonucleic acid (dsRNA) targeting a hepcidin antimicrobial peptide (HAMP) and/or HAMP-related gene, and methods of using the dsRNA to inhibit expression of HAMP and/or HAMP-related genes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 61/499,516, filed Jun. 21, 2011, and claims the benefit of U.S. Provisional Application Ser. No. 61/569,054, filed Dec. 9, 2011; each of which are incorporated herein by reference, in their entirety, for all purposes.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically as a text file named ______.txt, created on ______, 2012, with a size of ______ bytes. The sequence listing is incorporated by reference.

FIELD

The disclosure relates to double-stranded ribonucleic acid (dsRNA) targeting HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1, and methods of using dsRNA to inhibit expression of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1.

BACKGROUND

The discovery of the hepcidin peptide and characterization of its gene, HAMP, has led to the revision of previous models for the regulation of iron homeostasis and the realization that the liver plays a key role in determining iron absorption from the gut and iron release from recycling and storage sites. In summary, the hepcidin model proposes that the rate of iron efflux into the plasma depends primarily on the plasma level of hepcidin; when iron levels are high the synthesis of hepcidin increases and the release of iron from enterocytes and macrophages is diminished. Conversely when iron stores drop, the synthesis of hepcidin is down-regulated and these cells release more iron. Hepcidin directly binds to ferroportin and decreases its functional activity by causing it to be internalized from the cell surface and degraded.

Hepcidin provides a unifying hypothesis to explain the behavior of iron in two diverse but common clinical conditions, the anemia of chronic disease and both HFE and non-HFE haemochromatosis. The pathophysiology of hepcidin has been sufficiently elucidated to offer promise of therapeutic intervention in both of these situations. Administering either hepcidin or an agonist could treat haemochromatosis, where the secretion of hepcidin is abnormally low.

The anemia of inflammation, commonly observed in patients with chronic infections, malignancy, trauma, and inflammatory disorders, is a well-known clinical entity. Until recently, little was understood about its pathogenesis. It now appears that the inflammatory cytokine IL-6 induces production of hepcidin, an iron-regulatory hormone that may be responsible for most or all of the features of this disorder. (Andrews N C. J Clin Invest. 2004 May 1; 113(9): 1251-1253). As such, down regulation of hepcidin in anemic patients will lead to a reduction in inflammation associated with such anemia.

Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.

The following publications disclose dsRNA (siRNA) targeting the HAMP gene and are herein incorporated by reference for all purposes: WO 2008/036933 (International application no. PCT/US2007/079212, filed Sep. 21, 2007); US 2009-0209478 (U.S. patent application Ser. No. 11/859,288, filed Sep. 21, 2007); US 2010-0204307 (U.S. patent application Ser. No. 12/757,497, filed Apr. 9, 2010); US 2011-0269823 (U.S. patent application Ser. No. 13/184,087, filed Jul. 15, 2011).

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the HAMP1 mRNA levels in mouse liver following various dosages of siRNA and the serum iron concentration (μg/dL) following various dosages of siRNA in mice.

FIG. 2 shows the HAMP mRNA levels in liver following siRNA administration as well as the serum iron concentration (μg/dL) and the HAMP serum protein concentration (mg/mL) following siRNA administration to non-human primates.

FIG. 3 shows the HAMP1 and TFR2 mRNA levels in mouse liver following various dosages of siRNA and the percent (%) transferrin saturation following various dosages of siRNA.

FIG. 4 shows the HAMP1 and TFR2 mRNA levels in mouse liver following administration of siRNA and the percent (%) transferrin saturation over a 30 day time course.

FIG. 5 shows the HAMP1 and TFR2 mRNA levels in rat liver following administration of siRNA. FIG. 5 also shows the serum iron and Hb concentrations in rats at various time points.

FIG. 6 shows the level of HAMP mRNA reduction in the liver of each animal following siRNA administration, compared to PBS controls.

FIG. 7 shows the level of TFR2 mRNA reduction in the liver of each animal following siRNA administration, compared to PBS controls.

FIG. 8 shows that serum iron concentration was increased in each animal after 1 mg/kg AD-52590 siRNA administration.

FIG. 9 shows that the HAMP serum protein concentration was decreased in each animal following 1 mg/kg AD-52590 siRNA administration.

FIG. 10 shows combinatorial use of dsRNAs targeting different HAMP-related mRNAs (HFE and TFR2) in vivo. FIG. 10A shows relative mRNA levels for HFE (left bar), TFR2 (middle bar), and HAMP (right bar) for each group. FIG. 10B shows UIBC (μg/dL) for each group. FIG. 10C shows the percent transferring saturation for each group. FIG. 10D shows serum iron concentration (μg/dL) for each group.

SUMMARY

Disclosed herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of hepcidin antimicrobial peptide (HAMP), wherein said dsRNA is selected from the dsRNAs listed in Table 2, 3, 4, or 5 with a start position of 379, 380, 382, or 385. In some aspects, the dsRNA consists of a dsRNA listed in Table 2, 3, 4, or 5 with a start position of 382.

Also described herein is a dsRNA for inhibiting expression of HAMP, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a HAMP mRNA transcript, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 2, 3, 4, or 5.

In some aspects, the region of complementarity is at least 17 nucleotides in length. In some aspects, the region of complementarity is between 19 and 21 nucleotides in length. In some aspects, the region of complementarity is 19 nucleotides in length. In some aspects, the region of complementarity consists of one of the antisense strand sequences of Table 2, 3, 4, or 5.

In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 2, 3, 4, or 5. In some aspects, the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the sense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the sense strand comprises one of the sense strand sequences of Table 2, 3, 4, or 5. In some aspects, the antisense strand comprises one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the sense strand comprises one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand comprises one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the sense strand consists of one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand consists of one of the antisense strand sequences of Table 2, 3, 4, or 5. In some aspects, the dsRNA mediates degradation of HAMP mRNA.

In some aspects, said dsRNA further comprises at least one modified nucleotide. In some aspects, at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. In some aspects, said modified nucleotide is chosen from the group consisting of: a 2′-fluoro modified nucleotide, a 2′-fluoro modified nucleoside, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

In some aspects, each strand is no more than 30 nucleotides in length. In some aspects, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In some aspects, at least one strand comprises a 3′ overhang of at least 2 nucleotides. In some aspects, each strand comprises a 3′ overhang of 2 nucleotides.

In some aspects, a dsRNA described above further comprises a ligand. In some aspects, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA. In some aspects, the dsRNA further comprises an N-Acetyl-Galactosamine (GalNac) conjugate.

In some aspects, a dsRNA described above is formulated in a nucleic acid lipid particle formulation. In some aspects, the nucleic acid lipid particle formulation is selected from Table A. In some aspects, the nucleic acid lipid particle formulation comprises MC3.

Also described herein is a cell comprising a dsRNA described above.

Also described herein is a vector encoding at least one strand of a dsRNA described above.

Also described herein is a cell comprising a vector described above.

Also described herein is a pharmaceutical composition for inhibiting expression of a HAMP gene comprising a dsRNA described above. In some aspects, the composition further comprises a lipid formulation. In some aspects, the lipid formulation is a nucleic acid lipid particle formulation.

Also described herein is a dsRNA for inhibiting expression of hemojuvelin (HFE2), wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a HFE2 mRNA transcript, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10A.

In some aspects, the region of complementarity is at least 17 nucleotides in length. In some aspects, the region of complementarity is between 19 and 21 nucleotides in length. In some aspects, the region of complementarity is 19 nucleotides in length. In some aspects, the region of complementarity consists of one of the antisense strand sequences of Table 10A.

In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10A. In some aspects, the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10A. In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10A and the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10A. In some aspects, the sense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the sense strand sequences of Table 10A and the antisense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the antisense strand sequences of Table 10A. In some aspects, the sense strand comprises one of the sense strand sequences of Table 10A. In some aspects, the antisense strand comprises one of the antisense strand sequences of Table 10A. In some aspects, the sense strand comprises one of the sense strand sequences of Table 10A and the antisense strand comprises one of the antisense strand sequences of Table 10A. In some aspects, the sense strand consists of one of the sense strand sequences of Table 10A and the antisense strand consists of one of the antisense strand sequences of Table 10A. In some aspects, the dsRNA mediates degradation of HFE2 mRNA.

In some aspects, said dsRNA further comprises at least one modified nucleotide. In some aspects, at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. In some aspects, said modified nucleotide is chosen from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

In some aspects, each strand is no more than 30 nucleotides in length. In some aspects, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In some aspects, at least one strand comprises a 3′ overhang of at least 2 nucleotides. In some aspects, each strand comprises a 3′ overhang of 2 nucleotides.

In some aspects, a dsRNA described above further comprises a ligand. In some aspects, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA. In some aspects, a dsRNA described above further comprises a GalNac conjugate.

In some aspects, the dsRNA is formulated in a nucleic acid lipid particle formulation. In some aspects, the nucleic acid lipid particle formulation is selected from Table A. In some aspects, the nucleic acid lipid particle formulation comprises MC3.

Also described herein is a cell comprising a dsRNA described above.

Also described herein is a vector encoding at least one strand of a dsRNA described above.

Also described herein is a cell comprising a vector described above.

Also described herein is a pharmaceutical composition for inhibiting expression of a HFE2 gene comprising a dsRNA described above. In some aspects, the composition further comprises a lipid formulation. In some aspects, the lipid formulation is a nucleic acid lipid particle formulation.

Also described herein is a dsRNA for inhibiting expression of transferrin receptor 2 (TFR2), wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a TFR2 mRNA transcript, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10B or 13.

In some aspects, the region of complementarity is at least 17 nucleotides in length. In some aspects, the region of complementarity is between 19 and 21 nucleotides in length. In some aspects, the region of complementarity is 19 nucleotides in length. In some aspects, the region of complementarity consists of one of the antisense strand sequences of Table 10B or 13.

In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10B or 13. In some aspects, the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10B or 13. In some aspects, the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10B or 13 and the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10B or 13. In some aspects, the sense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the sense strand sequences of Table 10B or 13 and the antisense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the antisense strand sequences of Table 10B or 13. In some aspects, sense strand comprises one of the sense strand sequences of Table 10B or 13. In some aspects, the antisense strand comprises one of the antisense strand sequences of Table 10B or 13. In some aspects, the sense strand comprises one of the sense strand sequences of Table 10B or 13 and the antisense strand comprises one of the antisense strand sequences of Table 10B or 13. In some aspects, the sense strand consists of one of the sense strand sequences of Table 10B or 13 and the antisense strand consists of one of the antisense strand sequences of Table 10B or 13. In some aspects, the dsRNA mediates degradation of TFR2 mRNA.

In some aspects, said dsRNA further comprises at least one modified nucleotide. In some aspects, at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. In some aspects, said modified nucleotide is chosen from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

In some aspects, each strand is no more than 30 nucleotides in length. In some aspects, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In some aspects, at least one strand comprises a 3′ overhang of at least 2 nucleotides. In some aspects, each strand comprises a 3 overhang of 2 nucleotides.

In some aspects, a dsRNA described above further comprises a ligand. In some aspects, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA. In some aspects, a dsRNA described above further comprises a GalNac conjugate.

In some aspects, the dsRNA is formulated in a nucleic acid lipid particle formulation. In some aspects, the nucleic acid lipid particle formulation is selected from Table A. In some aspects, the nucleic acid lipid particle formulation comprises MC3.

Also described herein is a cell comprising a dsRNA described above.

Also described herein is a vector encoding at least one strand of a dsRNA described above.

Also described herein is a cell comprising a vector described above.

Also described herein is a pharmaceutical composition for inhibiting expression of a TFR2 gene comprising a dsRNA described above. In some aspects, the composition further comprises a lipid formulation. In some aspects, the lipid formulation is a nucleic acid lipid particle formulation.

Also described herein is a composition comprising a first dsRNA for inhibiting expression of a HAMP gene and a second dsRNA for inhibiting expression of an HFE2 gene, wherein the first dsRNA comprises a first sense strand and an first antisense strand, the first antisense strand comprising a region of complementarity to a HAMP mRNA transcript, wherein the first antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 2, 3, 4, or 5; and wherein the second dsRNA comprises a second sense strand and a second antisense strand, the second antisense strand comprising a region of complementarity to a HFE2 mRNA transcript, wherein the second antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10A.

Also described herein is a composition comprising a first dsRNA for inhibiting expression of a HAMP gene and a second dsRNA for inhibiting expression of an TFR2 gene, wherein said first dsRNA comprises a first sense strand and a first antisense strand, the first antisense strand comprising a region of complementarity to a HAMP mRNA transcript, wherein the first antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 2, 3, 4, or 5; and wherein said second dsRNA comprises a second sense strand and a second antisense strand, the second antisense strand comprising a region of complementarity to a TFR2 mRNA transcript, wherein the second antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10B or 13.

Also described herein is a composition comprising a first dsRNA for inhibiting expression of a TFR2 gene and a second dsRNA for inhibiting expression of a HFE2 gene, wherein said first dsRNA comprises a first sense strand and a first antisense strand, the first antisense strand comprising a region of complementarity to a TFR2 mRNA transcript, wherein the first antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10B or 13; and wherein said second dsRNA comprises a second sense strand and a second antisense strand, the second antisense strand comprising a region of complementarity to a HFE2 mRNA transcript, wherein the second antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10A.

Also described herein is a composition comprising a plurality of dsRNAs selected from the dsRNAs described above.

Also described herein is a method of inhibiting HAMP expression in a cell, the method comprising: (a) introducing into the cell a dsRNA described above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a HAMP gene, thereby inhibiting expression of the HAMP gene in the cell. In some aspects, the HAMP expression is inhibited by at least 30%. In some aspects, the HAMP expression is inhibited by at least 80%.

Also described herein is a method of treating a disorder associated with HAMP expression comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA described above.

In some aspects, the subject has anemia. In some aspects, the subject has refractory anemia. In some aspects, the subject has anemia of chronic disease (ACD). In some aspects, the subject has iron-restricted erythropoiesis. In some aspects, the subject is a human.

In some aspects, the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.

In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.

Also described herein is a method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of a HAMP dsRNA described above.

In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is administered intravenously. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.

In some aspects, the subject is a primate or a rodent. In some aspects, the subject is a human.

In some aspects, the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.

In some aspects, the subject has fatigue, shortness of breath, headache, dizziness, or pale skin. In some aspects, the subject has reduced iron levels compared to a subject without anemia. In some aspects, the subject has haemoglobin (Hb) levels <9 g/dL. In some aspects, the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA). In some aspects, the subject has reduced renal erythropoietin (EPO) synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD. In some aspects, the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD. In some aspects, the subject has a reticulocyte Hb of <28 pg. In some aspects, the subject has >10% hypochromic red blood cells (RBCs). In some aspects, the method further comprises determining the complete blood count (CBC), serum iron concentration, Transferrin (Tf) saturation, or ferritin levels of the subject.

In some aspects, administering results in an increase in iron levels in the subject. In some aspects, administering results in a 2-fold increase in iron levels in the subject. In some aspects, administering results in an increase in Tf saturation in the subject.

In some aspects, the method further comprises determining the iron level in the subject. In some aspects, the method further comprises administering intravenous iron or ESAs to the subject.

Also described herein is a method of inhibiting HFE2 expression in a cell, the method comprising: (a) introducing into the cell a dsRNA described above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a HFE2 gene, thereby inhibiting expression of the HFE2 gene in the cell. In some aspects, the HFE2 expression is inhibited by at least 30%. In some aspects, the HFE2 expression is inhibited by at least 80%.

Also described herein is a method of treating a disorder associated with HFE2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA described above.

In some aspects, the subject has anemia. In some aspects, the subject has refractory anemia. In some aspects, the subject has anemia of chronic disease (ACD). In some aspects, the subject has iron-restricted erythropoiesis. In some aspects, the subject is a human.

In some aspects, the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.

In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.

Also described herein is a method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of a HFE2 dsRNA described above.

In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is administered intravenously. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.

In some aspects, the subject is a primate or a rodent. In some aspects, the subject is a human.

In some aspects, the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.

In some aspects, the subject has fatigue, shortness of breath, headache, dizziness, or pale skin. In some aspects, the subject has reduced iron levels compared to a subject without anemia. In some aspects, the subject has haemoglobin (Hb) levels <9 g/dL. In some aspects, the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA). In some aspects, the subject has reduced renal erythropoietin (EPO) synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD. In some aspects, the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD. In some aspects, the subject has a reticulocyte Hb of <28 pg. In some aspects, the subject has >10% hypochromic red blood cells (RBCs). In some aspects, the method further comprises determining the complete blood count (CBC), serum iron concentration. Transferrin (Tf) saturation, or ferritin levels of the subject.

In some aspects, administering results in an increase in iron levels in the subject. In some aspects, administering results in a 2-fold increase in iron levels in the subject. In some aspects, administering results in an increase in Tf saturation in the subject.

In some aspects, the method further comprises determining the iron level in the subject. In some aspects, the method further comprises administering intravenous iron or ESAs to the subject.

Also described herein is a method of inhibiting TFR2 expression in a cell, the method comprising: (a) introducing into the cell a dsRNA described above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TFR2 gene, thereby inhibiting expression of the TFR2 gene in the cell. In some aspects, the TFR2 expression is inhibited by at least 30%. In some aspects, the TFR2 expression is inhibited by at least 80%.

Also described herein is a method of treating a disorder associated with TFR2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA described above.

In some aspects, the subject has anemia. In some aspects, the subject has refractory anemia. In some aspects, the subject has anemia of chronic disease (ACD). In some aspects, the subject has iron-restricted erythropoiesis. In some aspects, the subject is a human.

In some aspects, the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.

In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.

Also described herein is a method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of a TFR2 dsRNA described above.

In some aspects, the dsRNA is lipid formulated. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the dsRNA is administered intravenously. In some aspects, the dsRNA is lipid formulated in a formulation selected from Table A. In some aspects, the dsRNA is conjugated to GalNac. In some aspects, the dsRNA is conjugated to GalNac and administered subcutaneously. In some aspects, the dsRNA is administered subcutaneously.

In some aspects, the subject is a primate or a rodent. In some aspects, the subject is a human.

In some aspects, the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.

In some aspects, the subject has fatigue, shortness of breath, headache, dizziness, or pale skin. In some aspects, the subject has reduced iron levels compared to a subject without anemia. In some aspects, the subject has haemoglobin (Hb) levels <9 g/dL. In some aspects, the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA). In some aspects, the subject has reduced renal erythropoietin (EPO) synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD. In some aspects, the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD. In some aspects, the subject has a reticulocyte Hb of <28 pg. In some aspects, the subject has >10% hypochromic red blood cells (RBCs). In some aspects, the method further comprises determining the complete blood count (CBC), serum iron concentration, Transferrin (Tf) saturation, or ferritin levels of the subject.

In some aspects, administering results in an increase in iron levels in the subject. In some aspects, administering results in a 2-fold increase in iron levels in the subject. In some aspects, administering results in an increase in Tf saturation in the subject.

In some aspects, the method further comprises determining the iron level in the subject. In some aspects, the method further comprises administering intravenous iron or ESAs to the subject.

Also described herein is a method of inhibiting HAMP, HFE2, and/or TFR2 expression in a cell, the method comprising: (a) introducing into the cell a plurality of dsRNAs selected from the dsRNAs described above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a HAMP, HFE2, and/or TFR2 gene, thereby inhibiting expression of the HAMP, HFE2, and/or TFR2 gene in the cell.

In some aspects, the plurality of dsRNAs are introduced simultaneously. In some aspects, the plurality of dsRNAs are introduced concurrently. In some aspects, the plurality of dsRNAs are introduced individually. In some aspects, the plurality of dsRNAs are introduced together. In some aspects, the expression is inhibited by at least 30%. In some aspects, the expression is inhibited by at least 80%.

Also described herein is a method of treating a disorder associated with HAMP, HFE2, and/or TFR2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of a plurality of dsRNAs selected from the dsRNAs described above.

In some aspects, the plurality of dsRNAs are administered to the subject simultaneously. In some aspects, the plurality of dsRNAs are administered to the subject concurrently. In some aspects, the plurality of dsRNAs are administered to the subject individually. In some aspects, the plurality of dsRNAs are administered to the subject together.

In some aspects, the subject has anemia. In some aspects, the subject has refractory anemia. In some aspects, the subject has anemia of chronic disease (ACD). In some aspects, the subject has iron-restricted erythropoiesis. In some aspects, the subject is a human. In some aspects, the plurality is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.

Also described herein is a method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of a plurality of dsRNAs selected from the dsRNAs described above.

In some aspects, the plurality of dsRNAs are administered to the subject simultaneously. In some aspects, the plurality of dsRNAs are administered to the subject concurrently. In some aspects, the plurality of dsRNAs are administered to the subject individually. In some aspects, the plurality of dsRNAs are administered to the subject together.

In some aspects, the plurality is lipid formulated. In some aspects, the plurality is lipid formulated in a nucleic acid lipid particle formulation. In some aspects, the plurality is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously. In some aspects, the plurality is administered intravenously. In some aspects, the plurality is lipid formulated in a formulation selected from Table A. In some aspects, the plurality is conjugated to GalNac. In some aspects, the plurality is conjugated to GalNac and administered subcutaneously. In some aspects, the plurality is administered subcutaneously.

In some aspects, the subject is a primate or a rodent. In some aspects, the subject is a human.

In some aspects, the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.

In some aspects, the subject has fatigue, shortness of breath, headache, dizziness, or pale skin. In some aspects, the subject has reduced iron levels compared to a subject without anemia. In some aspects, the subject has haemoglobin (Hb) levels <9 g/dL. In some aspects, the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA). In some aspects, the subject has reduced renal erythropoietin (EPO) synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD. In some aspects, the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD. In some aspects, the subject has a reticulocyte Hb of <28 pg. In some aspects, the subject has >10% hypochromic red blood cells (RBCs). In some aspects, the method further comprises determining the complete blood count (CBC), serum iron concentration, Transferrin (Tf) saturation, or ferritin levels of the subject.

In some aspects, administering results in an increase in iron levels in the subject. In some aspects, administering results in a 2-fold increase in iron levels in the subject. In some aspects, administering results in an increase in Tf saturation in the subject.

In some aspects, the method further comprises determining the iron level in the subject. In some aspects, the method further comprises administering intravenous iron or ESAs to the subject.

DETAILED DESCRIPTION

The details of one or more embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the description and the drawings, and from the claims.

Provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of HAMP in a cell or a mammal where the dsRNA targets HAMP. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of HAMP. A HAMP dsRNA directs the sequence-specific degradation of HAMP mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of HFE2 in a cell or a mammal where the dsRNA targets HFE2. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of HFE2. A HFE2 dsRNA directs the sequence-specific degradation of HFE2 mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of HFE in a cell or a mammal where the dsRNA targets HFE. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of HFE. A HFE dsRNA directs the sequence-specific degradation of HFE mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of TFR2 in a cell or a mammal where the dsRNA targets TFR2. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of TFR2. A TFR2 dsRNA directs the sequence-specific degradation of TFR2 mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of BMPR1a in a cell or a mammal where the dsRNA targets BMPR1a. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of BMPR1a. A BMPR1a dsRNA directs the sequence-specific degradation of BMPR1a mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of SMAD4 in a cell or a mammal where the dsRNA targets SMAD4. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of SMAD4. A SMAD4 dsRNA directs the sequence-specific degradation of SMAD4 mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of IL6R in a cell or a mammal where the dsRNA targets IL6R. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of IL6R. An IL6R dsRNA directs the sequence-specific degradation of IL6R mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of BMP6 in a cell or a mammal where the dsRNA targets BMP6. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of BMP6. A BMP6 dsRNA directs the sequence-specific degradation of BMP6 mRNA.

Also provided herein are dsRNAs and methods of using the dsRNAs for inhibiting the expression of NEO1 in a cell or a mammal where the dsRNA targets NEO1. Also provided are compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of NEO1. A NEO1 dsRNA directs the sequence-specific degradation of NEO1 mRNA.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.

As used herein, “HAMP” refers to the hepcidin antimicrobial peptide gene, transcript, or protein (also known as LEAP). A human mRNA sequence for HAMP is Genbank accession NM_(—)021175.2, included below as SEQ ID NO:1. Other examples of mammalian HAMP sequences are shown in Table B.

As used herein. “HFE2” refers to hemojuvelin gene, transcript, or protein. Examples of mammalian HFE2 sequences are shown in Table B.

As used herein. “TFR2” refers to transferrin receptor 2 gene, transcript, or protein. Examples of mammalian TFR2 sequences are shown in Table B.

As used herein, “HFE” refers to hemochromatosis gene, transcript, or protein. Examples of mammalian HFE sequences are shown in Table B.

As used herein, “BMPR1a” refers to bone morphogenetic protein receptor, type 1A gene, transcript, or protein. Examples of mammalian BMPR1a sequences are shown in Table B.

As used herein, “SMAD4” refers to SMAD family member 4 gene, transcript, or protein. Examples of mammalian SMAD4 sequences are shown in Table B.

As used herein, “IL6R” refers to interleukin 6 receptor gene, transcript, or protein. Examples of mammalian IL6R sequences are shown in Table B.

As used herein, “BMP6” refers to bone morphogenetic protein 6 gene, transcript, or protein. Examples of mammalian BMP6 sequences are shown in Table B.

As used herein, “NEO1” refers to neogenin homolog 1 gene, transcript, or protein. Examples of mammalian NEO1 sequences are shown in Table B.

As used herein, “HAMP-related” refers to a HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, transcript, or protein.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, including mRNA that is a product of RNA processing of a primary transcription product.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as complementary with respect to a second sequence herein, the two sequences can be fully complementary, or they may be “substantially complementary,” e.g., they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1) including a 5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, a polynucleotide is complementary to at least a part of a HAMP mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding HAMP.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. The term “siRNA” is also used herein to refer to a dsRNA as described above.

As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA which includes a region that is complementary, e.g., fully complementary or substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is complementary, e.g., fully or substantially complementary to a region of the antisense strand.

The term “start position” refers to a nucleotide position on the target mRNA where the 5′ most nucleotide of a dsRNA sense strand aligns with the nucleotide position on the target mRNA. For example, a dsRNA with a start position of 382 on NM_(—)021175.2 (SEQ ID NO:1) would include AD-11459 because position 382 on NM_(—)021175.2 (SEQ ID NO:1) is G and the sense sequence of AD-11459 is 5′-GAAcAuAGGucuuGGAAuAdTsdT-3′ (SEQ ID NO:XX), where G is the 5′ most nucleotide of the sense strand of AD-11459; thus G at position 382 on NM_(—)021175.2 (SEQ ID NO:1) is the start position of AD-11459.

As used herein, the term “nucleic acid lipid particle” includes the term “SNALP” and refers to a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as a dsRNA or a plasmid from which a dsRNA is transcribed. Nucleic acid lipid particles, e.g., SNALP are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and U.S. Ser. No. 61/045,228 filed on Apr. 15, 2008. These applications are hereby incorporated by reference.

“Introducing into a cell,” when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein or known in the art.

The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of” and the like in as far as they refer to a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, herein refer to the at least partial suppression of the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, as manifested by a reduction of the amount of mRNA which may be isolated from a first cell or group of cells in which a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R. BMP6, and/or NEO1 gene is transcribed and which has or have been treated such that the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression, e.g., the amount of protein encoded by a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g., apoptosis. In principle, HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a HAMP, HFE2, HFE, TFR2. BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide featured in the invention.

As used herein in the context of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.

As used herein, the phrases “effective amount” refers to an amount that provides a benefit in the treatment, prevention, or management of pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression or an overt symptom of pathological processes mediated by HAMP, HFE2, HFE, TFR2. BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression. The specific amount that is effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression, the patient's history and age, the stage of pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression, and the administration of other anti-pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression agents.

As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a pharmacologically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter. For example, a pharmacologically effective amount of a dsRNA targeting HAMP can reduce HAMP serum levels by at least 25%.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.

Double-Stranded Ribonucleic Acid (dsRNA)

As described in more detail herein, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene in a cell or mammal, where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, and where the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and where said dsRNA, upon contact with a cell expressing said HAMP, HFE2, HFE, TFR2. BMPR1a, SMAD4, IL6R. BMP6, and/or NEO1 gene, inhibits the expression of said HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene by at least 30% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene can be reduced by at least 30% when measured by an assay as described in the Examples below. For example, expression of a HAMP gene in cell culture, such as in Hep3B cells, can be assayed by measuring HAMP mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by ELISA assay. The dsRNA of the invention can further include one or more single-stranded nucleotide overhangs.

The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. The dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, the other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length. In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different dsRNAs are used in combination, the duplex lengths can be identical or can differ. In one embodiment, the antisense strand of the dsRNA is sufficiently complementary to a target mRNA (e.g., a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 mRNA) so as to cause cleavage of the target mRNA.

Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each is strand is 25-30 nucleotides in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ.

The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the antisense strand. The dsRNA can include a 3′ overhang of 2 nucleotides on both the sense and antisense strands.

A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In one embodiment, a HAMP gene is a human HAMP gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Tables 2, 3, 4, and 5, and the antisense strand is one of the antisense sequences of Tables 2, 3, 4, and 5. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 2, 3, 4, and 5 can readily be determined using the target sequence and the flanking HAMP sequence.

The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2, 3, 4, and 5, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Tables 2, 3, 4, and 5 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 2, 3, 4, and 5, and differing in their ability to inhibit the expression of a HAMP gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired HAMP target sequence can readily be made using the corresponding HAMP antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Tables 2, 3, 4, and 5 identify a site in a HAMP that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 2, 3, 4, and 5 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a HAMP gene.

In one embodiment, a HFE2 gene is a human HFE2 gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Table 10A, and the antisense strand is one of the antisense sequences of Table 10A. Alternative antisense agents that target elsewhere in the target sequence provided in Table 10A can readily be determined using the target sequence and the flanking HFE2 sequence.

In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 10A, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 10A minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 10A, and differing in their ability to inhibit the expression of a HFE2 gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired HFE2 target sequence can readily be made using the corresponding HFE2 antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Table 10A identify a site in a HFE2 that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 10A coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a HFE2 gene.

In one embodiment, a TFR2 gene is a human TFR2 gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Table 10B or 13, and the antisense strand is one of the antisense sequences of Table 10B or 13. Alternative antisense agents that target elsewhere in the target sequence provided in Table 10B or 13 can readily be determined using the target sequence and the flanking TFR2 sequence.

In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 10B or 13, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 10B or 13 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 10B or 13, and differing in their ability to inhibit the expression of a TFR2 gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired TFR2 target sequence can readily be made using the corresponding TFR2 antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Table 10B or 13 identify a site in a TFR2 that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 10B or 13 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a TFR2 gene.

In one embodiment, a SMAD4 gene is a human SMAD4 gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Table 15 or 16, and the antisense strand is one of the antisense sequences of Table 15 or 16. Alternative antisense agents that target elsewhere in the target sequence provided in Table 15 or 16 can readily be determined using the target sequence and the flanking SMAD4 sequence.

In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 15 or 16, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 15 or 16 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 15 or 16, and differing in their ability to inhibit the expression of a SMAD4 gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired SMAD4 target sequence can readily be made using the corresponding SMAD4 antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Table 15 or 16 identify a site in a SMAD4 that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 15 or 16 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a SMAD4 gene.

In one embodiment, a NEO gene is a human NEO1 gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Table 17 or 18, and the antisense strand is one of the antisense sequences of Table 17 or 18. Alternative antisense agents that target elsewhere in the target sequence provided in Table 17 or 18 can readily be determined using the target sequence and the flanking NEO1 sequence.

In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 17 or 18, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 17 or 18 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 17 or 18, and differing in their ability to inhibit the expression of a NEO1 gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired NEO1 target sequence can readily be made using the corresponding NEO1 antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Table 17 or 18 identify a site in a NEO1 that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 17 or 18 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a NEO1 gene.

In one embodiment, a BMP6 gene is a human BMP6 gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Table 21, and the antisense strand is one of the antisense sequences of Table 21. Alternative antisense agents that target elsewhere in the target sequence provided in Table 21 can readily be determined using the target sequence and the flanking BMP6 sequence.

In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Table 21, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Table 21 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 21, and differing in their ability to inhibit the expression of a BMP6 gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired BMP6 target sequence can readily be made using the corresponding BMP6 antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Table 21 identify a site in a BMP6 that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Table 21 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a BMP6 gene.

With regard to Tables 4, 10A, 10B, 13, 16, 18, and 21: It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention. “Unmodified version” refers to a sequence that does not include one or more chemical modifications, e.g., a 2′-O methyl group, a phosphorothioate, and/or a 2′-fluoro group. For example, included in the invention are unmodified versions of AD-47391, which targets HFE2. See Table 10A. Unmodified sense strand versions of AD-47391 include: AGAGUAGGGAAUCAUGGCUdTdT and AGAGUAGGGAAUCAUGGCU. Unmodified antisense strand versions of AD-47391 include: AGCCAUGAUUCCCUACUCUdTdT and AGCCAUGAUUCCCUACUCU. As another example, included in the invention are unmodified versions of AD-47826, which targets TFR2. See Table 10B. Unmodified sense strand versions of AD-47826 include: CAGGCAGCCAAACCUCAUUdTdT and CAGGCAGCCAAACCUCAUU. Unmodified antisense strand versions of AD-47826 include: AAUGAGGUUUGGCUGCCUG and AAUGAGGUUUGGCUGCCUGdTdT.

Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. The cleavage site on the target mRNA of a dsRNA can be determined using methods generally known to one of ordinary skill in the art, e.g., the 5′-RACE method described in Soutschek et al., Nature; 2004, Vol. 432, pp. 173-178 (which is herein incorporated by reference for all purposes). Included in the invention are dsRNA that cleave the RNA target at the same location as the dsRNA described in the tables herein.

The dsRNA featured in the invention can contain one or more mismatches to the target sequence. In one embodiment, the dsRNA featured in the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of a HAMP gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R. BMP6, and/or NEO1 gene is important, especially if the particular region of complementarity in a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is known to have polymorphic sequence variation within the population.

Modifications

In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference

Modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts. In some instances, dsRNAs can be made with “Light Fluoro” chemical modifications as follows: all pyrimidines (cytosine and uridine) in the sense strand can be replaced with corresponding 2′-Fluoro bases (2′ Fluoro C and 2′-Fluoro U). In the antisense strand, pyrimidines adjacent to (towards 5′ position) ribo A nucleoside can be replaced with their corresponding 2-Fluoro nucleosides.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other suitable dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of a dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331: and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Other embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F: O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃. SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other preferred modifications include 2′-methoxy(2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro(2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

dsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

Conjugates

Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330: Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In some embodiments of the compositions and methods of the invention, an dsRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated dsRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g. C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

(Formula XXIII), when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

In some embodiments, the conjugate or ligand described herein can be attached to an dsRNA oligonucleotide with various linkers that can be cleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular dsRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)— (SEQ ID NO: 13), where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In one embodiment, an dsRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of dsRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

(Formula XXX), when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):

wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;

Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);

R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within a dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region.

In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306: Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded dsRNAs

In another aspect, HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 dsRNA molecules are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. NatI. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. NatI. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.

For example, lentiviral vectors featured in the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors featured in the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.

Viral vectors can be derived from AV and AAV. In one embodiment, the dsRNA featured in the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

The promoter driving dsRNA expression in either a DNA plasmid or viral vector featured in the invention may be a eukaryotic RNA polymerase I (e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g., U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single HAMP, HFE2, HFE, TFR2. BMPR1a, SMAD4, IL6R. BMP6, and/or NEO1 gene or multiple HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 genes over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Pharmaceutical Compositions Containing dsRNA

In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene, such as pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.

The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 genes.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.0059 mg/kg, 0.01 mg/kg, 0.0295 mg/kg, 0.05 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.628 mg kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

In one embodiment, the dosage is between 0.01 and 0.2 mg/kg. For example, the dsRNA can be administered at a dose of 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08 mg/kg 0.09 mg/kg, 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg kg, or 0.20 mg/kg.

In one embodiment, the dosage is between 0.005 mg/kg and 1.628 mg/kg. For example, the dsRNA can be administered at a dose of 0.0059 mg/kg, 0.0295 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, or 1.628 mg/kg.

In one embodiment, the dosage is between 0.2 mg/kg and 1.5 mg kg. For example, the dsRNA can be administered at a dose of 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, or 1.5 mg/kg.

The dsRNA can be administered at a dose of 0.03 mg/kg, or 0.03, 0.1, 0.2, or 0.4 mg/kg.

The pharmaceutical composition may be administered once daily or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The effect of a single dose on HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 levels is long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals, or at not more than 5, 6, 7, 8, 9, or 10 week intervals.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Administration

The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration.

The dsRNA can be delivered in a manner to target a particular tissue.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the dsRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearoylphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(MI), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(MI), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(MI) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_(1215G), that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B 1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., a nucleic acid lipid particle, e.g., SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. Nucleic acid lipid particles typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). Nucleic acid lipid particles are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site).

The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. In some embodiments the lipid to dsRNA ratio can be about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1.

In general, the lipid-nucleic acid particle is suspended in a buffer, e.g., PBS, for administration. In one embodiment, the pH of the lipid formulated siRNA is between 6.8 and 7.8, e.g., 7.3 or 7.4. The osmolality can be, e.g., between 250 and 350 mOsm/kg, e.g., around 300, e.g., 298, 299, 300, 301, 302, 303, 304, or 305.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200 or Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Cl₆), or a PEG-distearyloxypropyl (C₁₈). Other examples of PEG conjugates include PEG-cDMA (N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine), mPEG2000-DMG (mPEG-dimyrystylglycerol (with an average molecular weight of 2,000) and PEG-C-DOMG (R-3-[(o-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxlpropyl-3-amine). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol % of the total lipid present in the particle.

LNP01

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary formulations are described in Table A.

TABLE A cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Cationic Mol % ratios Lipid Lipid:siRNA ratio SNALP DLinDMA DLinDMA/DPPC/Cholesterol/PEG-cDMA (57.1/7.1/34.4/1.4) lipid:siRNA ~7:1 S-XTC XTC XTC/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP05 XTC XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP06 XTC XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP07 XTC XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP08 XTC XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP09 XTC XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 ALN100 ALN100/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP11 MC3 MC-3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP12 C12-200 C12-200/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. ______ filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.

MC3 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), (e.g., DLin-M-C3-DMA) comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and International Application No. PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated by reference.

ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine) comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.

C12-200, i.e., Tech G1, comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For a nucleic acid lipid formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions.

Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199: Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335, Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92: Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more dsRNA compounds and (b) one or more anti-cytokine biologic agents which function by a non-RNAi mechanism. Examples of such biologics include, biologics that target IL1β (e.g., anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab, or certolizumab).

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Methods for Inhibiting Expression of a HAMP, HFE2, HFE, TFR2. BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 Gene

In yet another aspect, the invention provides a method for inhibiting the expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is silenced.

When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection.

Methods for Treating Diseases Caused by Expression of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 Gene

The invention relates in particular to the use of a dsRNA targeting HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 and compositions containing at least one such dsRNA for the treatment of a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1-mediated disorder or disease. For example, the compositions described herein can be used to treat anemia and other diseases associated with lowered iron levels.

Methods of Using dsRNAs Targeting HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1

In one aspect, the invention provides use of a siRNA for inhibiting the expression of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 in a mammal. The method includes administering a composition of the invention to the mammal such that expression of the target HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is decreased. In some embodiments, HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R. BMP6, and/or NEO1 expression is decreased for an extended duration, e.g., at least one week, two weeks, three weeks, or four weeks or longer. For example, in certain instances, expression of the HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a siRNA described herein. In some embodiments, the HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 60%, 70%, or 80% by administration of the siRNA. In some embodiments, the HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide.

The methods and compositions described herein can be used to treat diseases and conditions that can be modulated by down regulating HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression. For example, the compositions described herein can be used to treat anemia and other forms of iron imbalance such as refractory anemia, refractory anemia of chronic disease (ACD), iron-restricted erythropoiesis, and the pathological conditions associated with these disorders. In some aspects, ACD subjects are those who are refractory to ESAs and i.v. iron administration. In some embodiments, the method includes administering an effective amount of a siRNA disclosed herein to a patient having lower iron levels relative to a control patient.

Therefore, the invention also relates to the use of a siRNA for the treatment of a disorder or disease mediated by or related to HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression. For example, a siRNA is used for treatment of anemia.

The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression preferably results in an enhancement of iron mobilization in the mammal. In some embodiments, iron mobilization is enhanced by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.

The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression preferably results in an Hb increase in the mammal. In some embodiments, Hb is increased by at least 10%, 15%, 20%, 25%, 30° %, 40%, 50%, or 60%, or more, as compared to pretreatment levels.

The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression preferably results in a serum iron increase in the mammal. In some embodiments, serum iron is increased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.

The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression preferably results in a transderrin (Tf) saturation increase in the mammal. In some embodiments, Tf saturation is increased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.

The effect of the decreased HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene expression preferably results in decreased levels of HAMP in the mammal. In some embodiments, HAMP is decreased by at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60%, or more, as compared to pretreatment levels.

The method includes administering a siRNA to the subject to be treated. The subject to be treated is generally a subject in need thereof. When the subject to be treated is a mammal, such as a human, the composition can be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, and airway (aerosol) administration. In some embodiments, the compositions are administered by intravenous infusion or injection.

The method includes administering a siRNA, e.g., a dose sufficient to depress levels of HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 mRNA for at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days; and optionally, administering a second single dose of dsRNA, wherein the second single dose is administered at least 5, more preferably 7, 10, 14, 21, 25, 30 or 40 days after the first single dose is administered, thereby inhibiting the expression of the target gene in a subject.

In one embodiment, doses of siRNA are administered not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administrations can be maintained for one, two, three, or six months, or one year or longer.

In another embodiment, administration can be provided when Hb levels reach or drop lower than a predetermined minimal level, such as less than 8 g/dL, 9 g/dL, or 10 g/dL. In some aspects, administration is continued until Hb levels are >11 g/dL, e.g, 12 g/dL.

In another embodiment, administration can be provided when a patient presents with various known symptoms of disorders such as anemia. These can include fatigue, shortness of breath, headache, dizziness, or pale skin.

In another embodiment, administration can be provided when a patient is diagnosed with anemia via CBC.

In general, the siRNA does not activate the immune system, e.g., it does not increase cytokine levels, such as TNF-alpha or IFN-alpha levels. For example, when measured by an assay, such as an in vitro PBMC assay, such as described herein, the increase in levels of TNF-alpha or IFN-alpha, is less than 30%, 20%, or 10% of control cells treated with a control dsRNA, such as a dsRNA that does not target HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1.

In an aspect, a subject can be administered a therapeutic amount of siRNA, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The siRNA can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the siRNA can reduce HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.

Before administration of a full dose of the siRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or IFN-alpha) levels.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given siRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Additional Agents and Co-Administration

In further embodiments, administration of a siRNA is administered in combination an additional therapeutic agent. The siRNA and an additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

In one embodiment, the siRNA is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the siRNA and the additional therapeutic agent are administered at the same time.

In some aspects, the additional agent can include one or more Erythropoiesis-stimulating agents (ESAs). ESAs are generally known in the art. ESAs can include Erythropoietin (EPO), Epoetin alfa (Procrit/Epogen), Epoetin beta (NeoRecormon), Darbepoetin alfa (Aranesp), and Methoxy polyethylene glycol-epoetin beta (Micera). ESAs can be administered in various doses, e.g., 7,000 U/week to 30,000 U/week.

In some aspects, the additional agent can include intravenous iron. Iron can be administered in various doses known in the art.

In some aspects, two or more dsRNAs are co-administered to a subject. In one embodiment, a first dsRNA is administered to the patient, and then a second dsRNA is administered to the patient (or vice versa). In another embodiment, the first dsRNA and the second dsRNA are administered at the same time.

In some aspects, a HAMP dsRNA is co-administered with one or more dsRNAs selected from HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 dsRNAs.

In some aspects, a HFE2 dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1dsRNAs.

In some aspects, a HFE dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1dsRNAs.

In some aspects, a TFR2 dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1dsRNAs.

In some aspects, a BMPR1a dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, TFR2, SMAD4, IL6R, BMP6, and/or NEO1 dsRNAs.

In some aspects, a SMAD4 dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, TFR2, BMPR1a, IL6R. BMP6, and/or NEO1 dsRNAs.

In some aspects, an IL6R dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, BMP6, and/or NEO1dsRNAs.

In some aspects, a BMP6 dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or NEO1dsRNAs.

In some aspects, a NEO dsRNA is co-administered with one or more dsRNAs selected from HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or BMP6 dsRNAs.

In another aspect, the invention features, a method of instructing an end user, e.g., a caregiver or a subject, on how to administer a siRNA described herein. The method includes, optionally, providing the end user with one or more doses of the siRNA, and instructing the end user to administer the siRNA on a regimen described herein, thereby instructing the end user.

Identification of Subjects in Need of dsRNA Administration

In one aspect, the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of iron mobilization. The method includes administering to the patient a siRNA in an amount sufficient to increase the patient's iron mobilization.

In one aspect, the invention provides a method of treating a patient by selecting a patient on the basis that the patient is in need of increased Hb levels. Such a subject can have Hb levels of <9 g/dL. The method includes administering to the patient a siRNA in an amount sufficient to increase the patient's Hb levels. Typically target Hb levels are >11 g/dL, e.g., 11 g/dL or 12 g/dL.

In some aspects, a subject is identified as having anemia. In some aspects, a subject is identified as having a refractory form of anemia. In some aspects, a subject is identified as having ACD. Such subjects can be in need of administration of a dsRNA described herein. ACD can include a form of anemia wherein the subject is refractory to ESAs and/or i.v. iron administration. Typical clinical presentation of ACD includes fatigue, shortness of breadth, headache, dizziness, and/or pale skin. ACD can also be diagnosed via a CBC test, which is generally known in the art. ACD can also be diagnosed via serum iron levels, Tf saturation, and/or ferritin levels. ACD is typically diagnosed in certain settings such as subjects with CKD, cancer, chronic inflammatory diseases such as RA, or IRIDA. In some aspects, a subject with ACD has Hb levels of less than 9 g/dL. Such subjects typically become symptomatic for ACD.

CKD can result in reduced renal EPO synthesis, dietary hematinic deficiencies, blood loss, and/or elevated hepcidin levels. The elevation in hepcidin levels can be due to decreased renal excretion and/or low grade inflammation characterized by, e.g., interleukin (IL)-6.

In some aspects, a subject is identified as having iron-restricted erythropoiesis (IRE). Such subjects can be in need of administration of a dsRNA described herein. IRE can be assessed via reticulocyte Hb (CHr). Typically a result of <28 pg suggests IRE, where normal is in the range of 28-35 pg. IRE can also be assessed via percent (%) hypochromic RBCs. Typically a result of >10% suggests IRE, where 1-5% is generally considered normal.

A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering a siRNA. In addition, a test may be performed to determine a geneotype or phenotype. For example, a DNA test may be performed on a sample from the patient, e.g., a blood sample, to identify the relevant genotype and/or phenotype before a dsRNA is administered to the patient. In another embodiment, a test is performed to identify a related genotype and/or phenotype.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the dsRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim. Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.

Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1.

TABLE 1 Abbreviations Abbreviation Nucleotide(s) A adenosine-3′-phosphate C cytidine-3′-phosphate G guanosine-3′-phosphate U uridine-3′-phosphate N any nucleotide (G, A, C, or T) a 2′-O-methyladenosine-3′-phosphate c 2′-O-methylcytidine-3′-phosphate g 2′-O-methylguanosine-3′-phosphate u 2′-O-methyluridine-3′-phosphate T, dT 2′-deoxythymidine-3′-phosphate sT; sdT 2′-deoxy-thymidine-5′phosphate-phosphorothioate Af 2′-fluoroadenosine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate

Example 2 HAMP siRNA Design

Transcripts

siRNA design was carried out to identify siRNAs targeting human, cynomolgus monkey (Macaca fascicularis; herein “cyno”), mouse, and rat HAMP transcripts annotated in the NCBI Gene database ncbi.nlm.nih.gov/gene website. In mouse, the HAMP gene is duplicated, yielding distinct HAMP1 and HAMP2 loci; duplex designs targeted only HAMP1. Design used the following transcripts from the NCBI RefSeq and GenBank collections: Human—NM_(—)021175.2 (SEQ ID NO:1); Cyno—EU076443.1; Mouse—NM_(—)032541.1; Rat—NM_(—)053469.1. Due to the short length of the HAMP transcripts and the high degree of primate/rodent HAMP sequence divergence, siRNA duplexes were designed in multiple separate batches. The separate batches are listed below and matched the various species as follows:

-   -   human and cyno HAMP, exactly;     -   only human HAMP, exactly;     -   human and cyno HAMP, with mismatches to HAMP in both species         allowed at sense-strand position 19 when a G or C HAMP         targeting-nucleotide was replaced with a U or A, i.e. “UA-swap”;     -   human and cyno HAMP, with exact match to human HAMP and         mismatches to cyno HAMP allowed at sense-strand positions 1, 2,         and 19, i.e. “mismatch-to-cyno”;     -   mouse HAMP1, exactly;     -   only rat HAMP, exactly.

All siRNA duplexes were designed that shared 100% identity with all listed human, cyno, mouse, or rat transcripts with the exception(s) of designated mismatched-to-target bases. Unless otherwise noted, duplexes themselves were 100% complementary and double-stranded.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand.

Table 2 provides the sequences of the sense and antisense strands of 42 duplexes targeting the 3′UTR of the human HAMP gene.

Table 3 provides the sequences of the sense and antisense strands of 47 duplexes targeting the CDS of the human HAMP gene.

Table 4 provides the sequences of the sense and antisense strands of the modified duplexes targeting the HAMP gene.

Table 5 provides the sequences of the sense and antisense strands of the unmodified version of the duplexes shown in Table 4.

The antisense-derived human/cyno, mouse, rat, UA-swap, and mismatch-to-cyno oligonucleotides shown in Tables 3-4 were synthesized and formed into duplexes.

In some instances the duplexes contained no chemical modifications (unmodified).

In some instances the duplexes contained modifications (modified). For example, some duplexes were made with “Light Fluoro” chemical modifications as follows: all pyrimidines (cytosine and uridine) in the sense strand were replaced with corresponding 2′-Fluoro bases (2′ Fluoro C and 2′-Fluoro U). In the antisense strand, pyrimidines adjacent to (towards 5′ position) ribo A nucleoside was replaced with their corresponding 2-Fluoro nucleosides.

Example 3 HAMP siRNA Screening

Cell Culture and Transfections:

Dual Luciferase System:

COS7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in DMEM (Gibco) supplemented with 10% FBS before being released from the plate by trypsinization. Cells were transfected with a psiCHECK2 vector (Promega) containing the human HAMP open reading frame (ORF). The ORF was introduced following the stop codon in the renilla luciferase sequence. Plasmid transfection was carried out by adding 19.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and 2.5 ng plasmid into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media containing ˜2×10⁴ COS7 cells were then added. Cells were incubated for three hours, after which the media was removed from the wells and replaced with 80 μl of complete growth media. Transfection of siRNA was accomplished out by preparing adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a new 96-well plate and incubated at room temperature for 15 minutes. The 20 μl volumes containing the lipoplexes were then added over the culture plates and incubated for 48 hours. Single dose experiments were performed at final concentrations of 10 nM and 0.1 nM. An additional concentration of 0.01 nM was performed for selected duplexes. Final duplex concentrations for dose response experiments were 10, 1.67, 0.278, 0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.

Endogenous System (Human):

For HAMP, HepG2 cells were used. HepG2 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in MEM (Gibco) supplemented with 10% FBS before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×10⁴ HepG2 cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at final concentrations of 10 nM and 0.1 nM. An additional concentration of 0.0 nM was performed for selected duplexes. Final duplex concentrations for dose response experiments were 10, 1.67, 0.278, 0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.

Endogenous System (Cynomolgus):

Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. Primary cynomolgus hepatocytes (M003055-P, Celsis) were thawed and prepared in InVitroGRO CP plating medium (Z99029, Celsis). 80 μl of complete growth media without antibiotic containing ˜2×10⁴ cynomolgus hepatocytes were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at final concentrations of 10 nM and 0.1 nM. Final duplex concentrations for dose response experiments were 10, 1.67, 0.278, 0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.

Dual Luciferase Assay (Promega Part E2980):

For cells transfected with the psiCHECK2 vector containing the human HAMP ORF, the Dual Luciferase assay was performed to measure reduction in HAMP levels. Forty-eight hours after transfection, the media was removed over the cells, and cells received 150 uL of a 1:1 mixture of complete growth medium and Dual-Glo Luciferase Reagent. As a control, these reagents were also added to empty wells; data derived from these samples were thus used as a blank measurement. Cells were then incubated for 30 minutes at room temperature on a shaker, protected from light. At this time, luminescence was determined using a SpectraMax M5 (Molecular Devices) with an integration time of 500 ms, and resulting data defined as the firefly luciferase signal. Following measurement, 75 μL of Dual-Glo Stop & Glo Reagent was added and the plates incubated in the dark at room temperature, without shaking. After an additional 10 minutes luminescence was again measured as above, and resulting data defined as the renilla luciferase signal. Data were background-subtracted, and the renilla values normalized to the firefly Luciferase values. Data were then expressed as percent mock-transfected or percent AD-1955.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR:

For human HAMP, 2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl HAMP TaqMan probe (Applied Biosystems cat #Hs00221783_ml) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). For cynomolgus HAMP, 2 μl of cDNA were added to a master mix containing 0.5 μl 18s TaqMan Probe (Applied Biosystems Cat #4319413 E), 0.1 μl 10× custom cynomolgus HAMP probe (Forward primer: CTCCGTTTTCCCACAACA (SEQ ID NO:XX); Reverse primer: CAGCACATCCCACACTTT (SEQ ID NO:XX); Probe: ACCCACTTCCCCATCTGCATT (SEQ ID NO:XX)), and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 100 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with 10 nM AD-1955, mock transfected, or to the average lowest dose.

Table 6 shows the HAMP single dose screening data of the modified duplexes using the dual luciferase assay. Data are expressed as a percent of mock or AD-1955.

Table 7 shows the HAMP single dose screening data of the unmodified duplexes using the human endogenous assay. Data are expressed as a percent of mock.

Table 8 shows the HAMP single dose screening data of the modified duplexes using the human endogenous assay. Data are expressed as a percent of mock.

Table 9 shows the HAMP dose response data of modified and unmodified duplexes using the dual luciferase assay. Cells used included HepG2 and Cyno primary hepatocytes.

Example 4 HFE2 siRNA Design

siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), mouse, and rat HFE2 transcripts annotated in the NCBI Gene database website noted above. There are at least 4 annotated human HFE2 transcripts and at least 3 annotated rhesus transcripts. Accordingly, we focused on the shortest annotated transcript for human, and the rhesus transcript which shared the greatest number of orthologous human exons, and designed on sequences held in common by the alternate transcripts. Design used the following transcripts from the NCBI RefSeq collection: Human—NM_(—)213652.3; Rhesus—XM_(—)001092987.1; Mouse—NM_(—)027126.4; Rat—NM_(—)001012080.1. Due to high primate/rodent sequence divergenge, siRNA duplexes were designed in two separate batches. The first batch matched human and rhesus; the second matched mouse and rat. All siRNA duplexes were designed that shared 100% identity with all listed human/rhesus or mouse/rat transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 4 or more Us or As in the seed region.

siRNA Sequence Selection

A total of 47 sense and 47 antisense derived human/rhesus, and 40 sense and 40 antisense derived mouse/rat siRNA oligos were synthesized and formed into duplexes.

Table 10A provides the sequences of the sense and antisense strands of the duplexes targeting the HFE2 gene.

Example 5 TFR2 siRNA Design

Transcripts

siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), mouse, and rat TFR2 transcripts annotated in the NCBI Gene database website noted above. Design used the following transcripts from the NCBI RefSeq collection: Human—NM_(—)003227.3, NM_(—)001206855.1; Rhesus—XM_(—)001113151.2; Mouse—NM_(—)015799.3; Rat—NM_(—)001105916.1. Due to high primate/rodent sequence divergenge, siRNA duplexes were designed in three separate batches. The first batch matched human and rhesus; the second matched human, rhesus, and mouse; the last batch matched mouse and rat. All siRNA duplexes were designed that shared 100% identity with all listed human/rhesus, human/rhesus/mouse, or mouse/rat transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region.

siRNA Sequence Selection

A total of 40 sense and 40 antisense derived human/rhesus, 5 sense and 5 antisense derived human/rhesus/mouse, and 45 sense and 45 antisense derived mouse/rat siRNA oligos were synthesized and formed into duplexes.

Table 10B provides the sequences of the sense and antisense strands of the duplexes targeting the TFR2 gene.

Example 6 HFE2 and TFR2 siRNA Screening

Cell Culture and Transfections:

Endogenous system (Human): For TFR2, HepG2 cells were used; Hep3b cells were used for HFE2. HepG2 and Hep3b cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in MEM (Gibco) supplemented with 10% FBS before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×10⁴ HepG2 cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at final concentrations of 100 nM and 0.1 nM. An additional concentration of 0.01 nM was performed for selected duplexes. Final duplex concentrations for dose response experiments were 10, 1.67, 0.278, 0.046, 0.0077, 0.0012, 0.0002, and 0.000035 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif. Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl HFE2 or TFR2 probes, and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). HFE2 and TFR2 probes were Applied Biosystems cat #Hs02378779_s1 and Hs00162690_m1, respectively. Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with 10 nM AD-1955, mock transfected, or to the average lowest dose.

Table 11 shows the HFE2 and TFR2 single dose screening data of the duplexes using the human endogenous assay.

Table 12 shows the HFE2 and TFR2 dose response data of the duplexes.

Example 7 HFE siRNA Design

Transcripts

siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), mouse, and rat HFE transcripts annotated in the NCBI Gene database website noted above. There are at least 9 annotated human HFE transcripts, at least 5 annotated rhesus transcripts, and at least 4 annotated rat transcripts. Accordingly, we focused on the shortest annotated transcripts for human, rhesus, and rat HFE, and designed on sequences held in common by the alternate transcripts. Design used the following transcripts from the NCBI RefSeq collection: Human—NM_(—)139006.2; Rhesus—XM_(—)001085598.2; Mouse—NM_(—)010424.4; Rat—NM_(—)001173435.1. Due to high primate/rodent sequence divergenge, siRNA duplexes were designed in two separate batches. The first batch matched human and rhesus; the second matched mouse and rat. All siRNA duplexes were designed that shared 100% identity with all listed human/rhesus or mouse/rat transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region.

siRNA Sequence Selection

A total of 46 sense and 46 antisense derived human/rhesus, and 24 sense and 24 antisense derived mouse/rat siRNA oligos are synthesized and formed into duplexes. The duplexes are screened using the methods described above. One or more duplexes are selected for further testing.

Example 8 BMPR1a siRNA Design

Transcripts

siRNA design was carried out to identify siRNAs targeting mouse and rat BMPR1A transcripts annotated in the NCBI Gene database website noted above. Design used the following transcripts from the NCBI RefSeq collection: Mouse—NM_(—)009758.4; Rat—NM_(—)030849.1. All siRNA duplexes were designed that shared 100% identity with all listed mouse/rat transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 4 or more Us or As in the seed region.

siRNA Sequence Selection

A total of 46 sense and 46 antisense derived mouse/rat siRNA oligos are synthesized and formed into duplexes. The duplexes are screened using the methods described above. One or more duplexes are selected for further testing.

Example 9 SMAD4 siRNA Design

Transcripts

siRNA design was carried out to identify siRNAs targeting human and mouse SMAD4 transcripts annotated in the NCBI Gene database website noted above. Design used the following transcripts from the NCBI RefSeq collection: Human—NM_(—)005359.5; Mouse—NM_(—)008540.2. All siRNA duplexes were designed that shared 100% identity with all listed human/mouse transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 4 or more Us or As in the seed region.

siRNA Sequence Selection

Tables 15-16 provide the sequences of the sense and antisense strands of the duplexes targeting SMAD4 mRNA at the indicated locations. Some duplexes were modified as indicated. These siRNA oligos were synthesized and formed into duplexes for further testing as described below.

Example 10 IL6R siRNA Design

Transcripts

siRNA design was carried out to identify siRNAs targeting mouse and rat IL6R transcripts annotated in the NCBI Gene database website noted above. Design used the following transcripts from the NCBI RefSeq collection: Mouse—NM_(—)010559.2; Rat—NM_(—)017020.3. All siRNA duplexes were designed that shared 100% identity with all listed mouse/rat transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position and had 2 or more Us or As in the seed region.

siRNA Sequence Selection

A total of 44 sense and 44 antisense derived mouse/rat siRNA oligos are synthesized and formed into duplexes. The duplexes are screened using the methods described above. One or more duplexes are selected for further testing.

Example 11 BMP6 siRNA Design

Transcripts

siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), mouse, and rat BMP6 transcripts annotated in the NCBI Gene database website noted above. Design used the following transcripts from the NCBI RefSeq collection: Human—NM_(—)001718.4; Rhesus—XM_(—)001085364.2; Mouse—NM_(—)007556.2; Rat—NM_(—)013107.1. Due to high primate/rodent sequence divergenge, siRNA duplexes were designed in three separate batches. The first batch matched human and rhesus; the second matched human, rhesus, and mouse: the last batch matched mouse and rat. All siRNA duplexes were designed that shared 100% identity with all listed human/rhesus, human/rhesus/mouse, or mouse/rat transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region.

siRNA Sequence Selection

Table 21 provides the sequences of the sense and antisense strands of the duplexes targeting BMP6 mRNA. Some duplexes were modified as indicated. These siRNA oligos were synthesized and formed into duplexes for further testing using the methods described herein.

Example 12 Neo1 siRNA Design

Transcripts

siRNA design was carried out to identify siRNAs targeting human and mouse NEO1 transcripts annotated in the NCBI Gene database website noted above. There are 2 annotated mouse NEO1 transcripts. Accordingly, we focused on the shortest annotated transcripts for mouse NEO1, and designed on sequences held in common by the alternate transcripts. Design used the following transcripts from the NCBI RefSeq collection: Human—NM_(—)002499.2; Mouse—NM_(—)001042752.1. All siRNA duplexes were designed that shared 100% identity with all listed human/mouse transcripts.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were selected that lacked repeats longer than 7 nucleotides. These siRNAs were used in comprehensive searches against the appropriate transcriptomes.

siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region.

siRNA Sequence Selection

Tables 17-18 provide the sequences of the sense and antisense strands of the duplexes targeting NEO1 mRNA at the indicated locations. Some duplexes were modified as indicated. These siRNA oligos were synthesized and formed into duplexes for further testing as described below.

Example 13 Activity of Murine siRNA In Vivo

The efficacy of one or more siRNAs described above is determined in mice, e.g., normal 10 week old 129s6/svEvTac mice using AD-1955 targeting luciferase as a control. The siRNAs are formulated as described herein and administered, e.g., through i.v. bolus at a dose of, e.g., 10 mg/kg. Forty eight hours after injection, the liver and serum samples are harvested. The liver mRNA levels of the target mRNA are determined by qRT-PCR using gene specific primers and serum iron levels are determined using Feroxcine (Randox Life Sciences) and Hitachi 717 instrument.

siRNAs that result in lowering of mRNA are selected for further evaluation.

Example 14 Activity of Murine Hepcidin siRNA In Vivo

The efficacy of an HAMP siRNA AD-10812 was determined in mice using AF-011 formulated control siRNA and PBS as controls. Each siRNA was formulated with AF-011. AF-011 is also known as LNP11 (See Table A above; MC-3/DSPC/Cholesterol/PEG-DMG (50/10/38.5/1.5); Lipid:siRNA 10:1)).

position in mouse access. # SEQ SEQ NM_03254 sense strand ID antisense strand ID duplex 1.1 sequence (5′-3′) NO sequence (5′-3′) NO name 245-263 uGcuGuAAcAAuucccAGuTsT ACUGGGAAUUGUuAcAGcATsT AD-10812

PBS and the siRNAs were administered at various dosages to the mice as shown in FIG. 1: 1 mg/kg, 0.3 mg/kg, 0.1 mg/kg, 0.03 mg/kg, 0.01 mg/kg, and 0.003 mg/kg. A single siRNA dose was administered to each mouse. After injection, liver and serum samples were harvested from the mice. The liver Hamp1 mRNA levels were determined by qRT-PCR using Hamp1 specific primers and serum iron levels were determined. FIG. 1 shows the HAMP1 mRNA levels in mouse liver following various dosages of siRNA and the serum iron concentration (μg/dL) following various dosages of siRNA.

Administration of AD-10812 HAMP siRNA to mice resulted in lowering of HAMP mRNA by >80% following a single dose. Administration of AD-10812 HAMP siRNA to mice resulted in an approximately 2-fold increase in serum iron following a single dose.

Example 15 Activity of Hepcidin siRNA in Nonhuman Primates (NHPs) In Vivo

The efficacy of an HAMP siRNA AD-11459 was determined in male cynomolgus monkeys using AF-011 siRNA as a control.

SEQ SEQ Duplex Start Sense Sense ID Antisense Antisense ID Target ID Position Name Sequence NO Name Sequence NO HAMP AD- 382 A-18280.2 GAAcAuAGGucuuG A-18304.1 uAuUCcAAGACCuAuGuUC 11459 GAAuAdTsdT dTsdT

Each siRNA was formulated with AF-011. The siRNAs were administered intravenously at a dose of 1 mg/kg via a 15 minute infusion. A single siRNA dose was administered to each monkey. After injection, liver and serum samples were harvested. Liver samples were taken at 48 hours (h) post-injection. Serum samples were taken at Day −9, Day −6, Day −3, 24 h post-injection, and 48 h post-injection. The liver Hamp mRNA levels were determined by qRT-PCR using Hamp specific primers and serum iron levels were determined. Serum HAMP protein levels were also determined. FIG. 2 shows the HAMP mRNA levels in liver following siRNA administration as well as the serum iron concentration (μg/dL) and the HAMP serum protein concentration (mg/mL) following siRNA administration.

Single administration of LNP-siRNA AD-11459 resulted in rapid reduction of hepcidin mRNA and protein levels and elevation of serum iron levels in NHPs.

Example 16 Silencing of Murine TFR2 Via siRNA In Vivo

The efficacy of TFR2 siRNA AD-47882 (see table below for sequences) was determined in C57BL6 mice using AF-011 siRNA and PBS as controls. Each siRNA was formulated with AF-011. PBS and the siRNAs were administered at various dosages to the mice as shown in FIG. 3: 1 mg/kg, 0.3 mg/kg, 0.1 mg/kg, and 0.03 mg/kg. After injection, liver and serum samples were harvested from the mice. The liver Hamp1 and TFR2 mRNA levels were determined by qRT-PCR using gene specific primers and transferrin saturation were determined at 48 hours post-injection. FIG. 3 shows the HAMP1 and TFR2 mRNA levels in mouse liver following various dosages of siRNA and the percent (%) transferrin saturation following various dosages of siRNA.

SEQ SEQ Duplex Sense ID Antisense ID Target ID Sequence NO Sequence NO TFR2 AD- ccAcGuGAuucuccu AGAAAGGAGAAUcACG 47882 uucudTsdT UGGdTsdT

Administration of AD-47882 siRNA to mice resulted in lowering of HAMP and TFR2 mRNA levels. Administration of AD-47882 siRNA to mice resulted in an increase in transferrin saturation.

Example 17 Silencing of Murine TFR2 Via siRNA In Vivo

The duration of TFR2 siRNA AD-47882 was determined in C57BL6 mice. The siRNAs were administered at in a single 0.3 mg/kg dose intravenously. Each siRNA was formulated with AF-011. After injection, liver and serum samples were harvested from the mice at various time points shown in FIG. 4. The liver Hamp1 and TFR2 mRNA levels were determined by qRT-PCR using gene specific primers and transferrin saturation were determined. FIG. 4 shows the HAMP1 and TFR2 mRNA levels in mouse liver following administration of siRNA and the percent (%) transferrin saturation over a 30 day time course.

Administration of AD-47882 siRNA to mice resulted in lowering of HAMP and TFR2 mRNA levels. Administration of AD-47882 siRNA to mice resulted in an increase in transferrin saturation.

Example 18 Silencing of Rat TFR2 Via siRNA In Vivo

The duration and efficacy of TFR2 siRNA AD-47882 was determined in male Lewis rats using the anemia of chronic disease (ACD) model described in Coccia et al., Exp. Hematology, 2001. Briefly, anemia was initiated in the rats with a single intraperaoneal (i.p.) injection of PG-APS (polymers from Group A Streptococci). The rats were then treated 3× per week with AD-47882 siRNA, AF-011 control siRNA, or saline control starting at day 21 post PG-APS. Each siRNA was formulated with AF-011. Serum and hematology parameters were measured biweekly and at 48 hours post final treatment. Serum samples were harvested from the rats at various time points as shown in FIG. 5. Liver mRNA measurement was taken at 48 hours post final treatment. FIG. 5 shows the HAMP1 and TFR2 mRNA levels in rat liver following administration of siRNA. FIG. 5 also shows the serum iron and Hb concentrations at various time points.

Administration of AD-47882 siRNA resulted in lowering of HAMP and TFR2 mRNA levels. Administration of AD-47882 siRNA resulted in an approximate 2× increase in serum iron upon treatment. Administration of AD-47882 siRNA resulted in an increase in Hb levels to 11-12 g/dL with treatment.

Example 19 TFR2 siRNA Selection and Screening

siRNA Sequence Selection

Table 13 provides the sequences of the sense and antisense strands of the duplexes targeting the TFR2 gene at the indicated locations (64 or 239). These siRNA oligos were synthesized and formed into duplexes.

Cell Culture and Transfections:

Endogenous system (Human): For TFR2, HepG2 cells were used. HepG2 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in MEM (Gibco) supplemented with 10% FBS before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×10⁴ HepG2 cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at final concentrations of 10 nM and 0.1 nM and 0.01 nM. An additional concentration of 0.01 nM was performed for selected duplexes.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.511 GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl TFR2 probes, and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). TFR2 probes were Applied Biosystems cat #Hs02378779_s1 and Hs00162690_m1, respectively. Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with 10 nM AD-1955, mock transfected, or to the average lowest dose.

Table 14 shows the TFR2 dose response data of the duplexes.

Example 20 Activity of TFR2 and HAMP siRNA in Non-Human Primates (NHPs) In Vivo

The efficacy of AD-52590, AD-51707, and AD-48141 was determined in separate cynomolgus monkeys (3 each) using PBS as a control. The sequence of AD-52590, AD-51707, and AD-48141 are shown below and in Table 4, 10B, and 13.

SEQ SEQ Duplex Start ID ID Target ID Position NO Sense Sequence NO Antisense Sequence TFR2 AD- 239 cAGGcAGcCAAAcCuCAuUdTsdT AAUGAGGUuUGGCUGcCugdTsdT 52590 TFR2 AD- 105 ccuucAAucAAAcccAGuudTsdT AACuGGGUuUGAuUGAAGGdTsdT 51707 HAMP AD- 382 GAAcAuAGGucuuGGAAuAdTdT UAuUCcAAGACCuAuGuUCdTdT 48141

Each siRNA was formulated with AF-011. The siRNAs were administered intravenously as indicated (0.1 mg/kg, 0.03 mg/kg, or 1 mg/kg) via a 15 minute infusion. A single siRNA dose was administered to each monkey. After injection, liver and serum samples were harvested. Liver biopsy samples were taken at 48 hours (h) post-injection. Serum samples were taken at Day −9, Day −6, Day −3, 24 h post-injection, and 48 h post-injection. The liver Hamp mRNA levels were determined by qRT-PCR using Hamp specific primers. The liver TFR2 mRNA levels were determined by qRT-PCR using TFR2 specific primers. Serum iron levels were determined and are shown in μg/dL. Serum HAMP protein levels were also determined and are shown in ng/mL.

FIG. 6 shows HAMP mRNA levels in the liver of each animal following siRNA administration, relative to PBS controls. FIG. 7 shows TFR2 mRNA levels in the liver of each animal following siRNA administration, relative to PBS controls. FIG. 8 shows that serum iron concentration was increased in each animal after 1 mg/kg AD-52590 siRNA administration. FIG. 9 shows that the HAMP serum protein concentration was decreased in each animal following 1 mg/kg AD-52590 siRNA administration.

Single administration of AD-52590 resulted in rapid reduction of hepcidin mRNA and protein levels, TFR2 mRNA levels, and elevation of serum iron levels in NHPs.

Example 21 NEO1 and SMAD4 Duplex Screening

Human/mouse cross-reactive Neo1 and Smad4 siRNAs were screened in primary mouse hepatocytes. Duplexes are shown in Tables 15, 16, 17, and 18.

Cell Culture and Transfections:

Freshly isolated primary mouse hepatocytes (PMH) were transfected by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of primary hepatocyte media containing ˜2×10⁴ PMH cells were then added to the siRNA mixture. Cells were incubated for either 24 prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12):

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl of mouse GAPDH TaqMan Probe (Applied Biosystems Cat #4352932E), 0.5 μl Neo1 or SMAD4 TaqMan probe (Applied Biosystems cat #Neo 1-Mm00476326_m1 or SMAD4 Mm03023996_m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections and each transfection was assayed in duplicate, unless otherwise noted.

To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose.

Table 19 shows the percent remaining mRNA remaining for each SMAD4 duplex tested at 0.1 nM and 10 nM. Controls were 10 nM AD-1955, mock transfected. Table 20 shows the percent remaining mRNA remaining for each NEO1 duplex tested at 0.1 nM and 100 nM. Controls were 10 nM AD-1955, mock transfected.

Example 22 In Vivo Combinatorial Use of dsRNAs Targeting HAMP-Related mRNAs

The efficacy of TFR2 siRNA AD-47882 and HFE siRNA AD-47320 (see table below for sequences) alone and in combination was determined in C57BL6 female mice using AF-011-Luc siRNA and PBS as controls. Each siRNA was formulated with AF-011. PBS and the siRNAs were administered at various (mg/kg) dosages to the mice as shown on the X-axis of each subfigure (A-D) of FIG. 10. 48 hours after injection, liver and serum samples were harvested from the mice.

SEQ SEQ Duplex Accession Sense ID Antisense ID Target ID Number Sequence NO Sequence NO HFE AD- NM_010424.4 uuuucuccAGuuAAG UGAACUuAACUGGAGA 47320 uucAdTsdT AAAdTsdT HFE Unmo NM_010424.4 UUUUCUCCAGUUA UGAACUUAACUGGAGA d AD- AGUUCA AAA 47320

The liver Hamp1, HFE, and TFR2 mRNA levels were determined by qRT-PCR using gene specific primers. Blood was processed into serum to measure serum iron, transferrin saturation, and UIBC. FIG. 10A shows the HAMP1, HFE, and TFR2 mRNA levels in mouse liver following various dosages of each siRNA group or PBS. FIGS. 10B-D shows serum iron concentration, transferrin saturation, and UIBC concentration in the serum of each group tested.

Example 23 Inhibition of HAMP in Humans

A human subject is treated with a siRNA targeted to a HAMP gene to inhibit expression of the HAMP gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

Example 24 Inhibition of HFE2 in Humans

A human subject is treated with a siRNA targeted to a gene to inhibit expression of the HFE2 gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

Example 25 Inhibition of HFE in Humans

A human subject is treated with a siRNA targeted to a gene to inhibit expression of the HFE gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, TFR2, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

Example 26 Inhibition of TFR2 in Humans

A human subject is treated with a siRNA targeted to a gene to inhibit expression of the TFR2 gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, BMPR1a, SMAD4, IL6R, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

Example 27 Inhibition of BMPR1a in Humans

A human subject is treated with a siRNA targeted to a gene to inhibit expression of the BMPR1a gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, TFR2, SMAD4, IL6R, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

Example 28 Inhibition of SMAD4 in Humans

A human subject is treated with a siRNA targeted to a gene to inhibit expression of the SMAD4 gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, TFR2, BMPR1a, IL6R, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

Example 29 Inhibition of IL6R in Humans

A human subject is treated with a siRNA targeted to a gene to inhibit expression of the IL6R gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, BMP6, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

Example 30 Inhibition of BMP6 in Humans

A human subject is treated with a siRNA targeted to a gene to inhibit expression of the BMP6 gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or NEO1 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

Example 31 Inhibition of NEO1 in Humans

A human subject is treated with a siRNA targeted to a gene to inhibit expression of the NEO1 gene to treat a condition. In some instances, one or more additional siRNAs are co-administered, e.g., an siRNA targeted to a HAMP, HFE2, HFE, TFR2, BMPR1a, SMAD4, IL6R, and/or BMP6 gene.

A subject in need of treatment is selected or identified.

The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an siRNA is administered to the subject. The siRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated. This measurement can be accompanied by a measurement of target gene expression in said subject, and/or the products of the successful siRNA-targeting of mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

Tables

TABLE B SEQ ID NO DESCRIPTION SEQUENCE  1 Human HAMP- gactgtcactcggtcccagacaccagagcaagctcaagacccagcagtgggacagcc NM_021175.2 agacagacggcacgatggcactgagctcccagatctgggccgcttgcctcctgctcc tcctcctcctcgccagcctgaccagtggctctgttttcccacaacagacgggacaac ttgcagagctgcaaccccaggacagagctggagccagggccagctggatgcccatgt tccagaggcgaaggaggcgagacacccacttccccatctgcattttctgctgcggct gctgtcatcgatcaaagtgtgggatgtgctgcaagacgtagaacctacctgccctgc ccccgtcccctcccttccttatttattcctgctgccccagaacataggtcttggaat aaaatggctggttctttcgttttccaaaaaa  2 Cyno HAMP- tcaagacctagcagtgggacagccagacagacggcacgatggcactgagctcccaga EU076443.1 tctgggccacttgcctcctcctccttctcctcctcgccagcctgaccagtggctccg ttttcccacaacagacgggacaacttgcagagctgcaacctcaggacagagctggag ccagggccagctggacgcccatgctccagaggcgaaggaggcgagacacccacttcc ccatctgcattttctgctgcggctgctgtcatcgatcaaagtgtgggatgtgctgca ggacgtagaaccttcctgccctgcccccatcccctcccttccttatttattcctgct gccccagaacacaggtcttggaataaaacggctgattcttttgttttcc  3 HAMP- agtccttagactgcacagcagaacagaaggcatgatggcactcagcactcggaccca NM_032541.1 ggctgcctgtctcctgcttctcctccttgccagcctgagcagcaccacctatctcca tcaacagatgagacagactacagagctgcagcctttgcacggggaagaaagcagggc agacattgcgataccaatgcagaagagaaggaagagagacaccaacttccccatctg catcttctgctgtaaatgctgtaacaattcccagtgtggtatctgttgcaaaacata gcctagagccacatcctgacctctctacacccctgcagcccctcaaccccattattt attcctgccctccccaccaatgaccttgaaataaagacgattttattttcaaaaaaa aaaaaaaaaaa  4 Rat HAMP- cacgagggcaggacagaaggcaagatggcactaagcactcggatccaggctgcctgt NM_053469.1 ctcctgcttctcctcctggccagcctgagcagcggtgcctatctccggcaacagacg agacagactacggctctgcagccttggcatggggcagaaagcaagactgatgacagt gcgctgctgatgctgaagcgaaggaagcgagacaccaacttccccatatgcctcttc tgctgtaaatgctgtaagaattcctcctgtggtctctgttgcataacatagagagcc aagagccttgtcctgacctctcaacacactgcctcccctccgccccattatttattc ctgtcctaccccagcaatgaccttg  5 Human HEFE2- accgtcaactcagtagccacctccctccctgctcagctgtccagtactctggccagc NM_213652.3 catatactcccccttccccccataccaaaccttctctggttccctgacctcagtgag acagcagccggcctggggacctgggggagacacggaggaccccctggctggagctga cccacagagtagggaatcatggctggagaattggatagcagagtaatgtttgacctc tggaaacactcaccatcatatttaagaacatgcaggaatgcattgatcagaaggtgt atcaggctgaggtggataatcttcctgtagcctttgaagatggttctatcaatggag gtgaccgacctgggggatccagtttgtcgattcaaactgctaaccctgggaaccatg tggagacccaagctgcctacattggcacaactataatcattcggcagacagctgggc agctctccttctccatcaaggtagcagaggatgtggccatggccttctcagctgaac aggacctgcagctctgtgttggggggtgccctccaagtcagcgactctctcgatcag agcgcaatcgtcggggagctataaccattgatactgccagacggctgtgcaagaaag ggcttccagtggaagatgcctacttccattcctgtgtctttgatgctttaatttctg gtgatcccaactttaccgtggcagctcaggcagcactggaggatgcccgagccttcc tgccagacttagagaagctgcatctcttcccctcagatgctggggttcctctttcct cagcaaccctcttagctccactcctttctgggctctttgttctgtggctttgcattc agtaaggggaccatcagccccattactagtttggaaatgatttggagatacagattg gcatagaagaatgtaaagaatcattaaaggaagcagggcctaggagacacgtgaaac aatgacattatccagagtcagatgaggctgcagtccagggttgaaattatcacagaa taaggattctgggcaaggttactgcattccggatctctgtggggctcttcaccaatt tttccagcctcatttatagtaaacaaattgttctaatccatttactgcagatttcac ccttataagtttagaggtcatgaaggttttaatgatcagtaaagatttaagggttga gatttttaagaggcaagagctgaaagcagaagacatgatcattagccataagaaact caaaggaggaagacataattagggaaagaagtctatttgatgaatatgtgtgtgtaa ggtatgttctgctttcttgattcaaaaatgaagcaggcattgtctagctcttaggtg aagggagtctctgcttttgaagaatggcacaggtaggacagaagtatcatccctacc ccctaactaatctgttattaaagctacaaattcttcacaccatcaaaaaaaaaaaaa aaaaaa  6 Rhesus HFE2- cttctctggctccctgacctcagtgagacagcagccggcctggggacctgggggaga XM_001092987 catggagaaagagacggaggaccccctggctggagctgacccacagagtagggaatc .1 atggctggagaattggatagcagagtaatgtttgacctctggaaacaccaaatttct tttttcagtcacttacagggcttccggtcaaaattcactaggtaggagggtcatcag ctgggaagaaccggcgcctggggaacctggctggataggtatgggggagcaaggcca gtcccctagtcccaggtcctcccatggcagccccccaactctaagcactctcactct cctgctgctcctctgtggacatgctcattcccaatgcaagatcctccgctgcaatgc tgagtatgtatcgtccactctgagccttagaggtggcggttcatcaggagcacttcg aggaggaggaggaggaggaggccggggtggaggggtgggctctggcggcctctgtcg agccctccgctcctatgcgctctgcactcggcgcaccgcccgcacctgccgtgggga cctcgccttccatccggcggtacatggcatcgaagacctgatgatccagcacaactg ctcgcgccagggccctacagcccctcccccgccccggggccccgcccttccaggcgc aggctccggcctccctgccccggacccttgtgactatgaaggccggtttccccggct gcatggtcgtcccccggggttcttgcattgcgcttccttcggggacccccatgtgcg cagcttccaccaccattttcacacatgccgtgtccaaggagcttggcctctactgga taacgacttcctccttgtccaagccaccagctcccccatggcgttgggggccaacgc taccgctacccggaagctcaccatcatatttaagaacatgcaggaatgcattgatca gaaggtctatcaggctgaggtggataatcttcctgcagcctttgaagatggttctgt caatggaggtgaccgacctgggggatccagtttgtcgattcaaactgctaaccctgg gaaccacgtggagatccaagctgcctacattggcacaactataatcattcggcagac agctgggcagctctccttctccatcaaggtagcagaggatgtggccatggccttctc agctgaacaggacctgcagctctgtgttggggggtgccctccaagtcagcgactctc tcgatcagagcgcagtcgtccgggagctataaccattgatactgccagacggctgtg taaggaagggcttccagtggaagatgcttacttccattcctgtgtctttgatgtttt aatttctggtgatcccaactttactgtggcagctcaggcagcactggaggatgcccg agccttcctgccagacttagataagctgcatctcttcccttcagatgctggggtttc tctttcctcagcaaccttcctagccccactcctttctgggctctttgttctgtggct ttgcattcagtaaggaagccatcagtcctattactagtttggaaatgatttggggat agagattggcatagaagaatgtaaacaatcattaaaggaagcagggcccagaagaca catgaaacaatgacatcatccagagtcagatgaggctgcagtccagggttgaaatga tcacagaataaggattctgggcaaggtttctgcattccagacctcttcgccaaattt tccagccccatttacagtaaacaaattgttctttccatttactgcagatttcaccct ataagcttagaggtcatgaaggttttaacaatcagtaaagacttaagggttgagatt tttaagaggcaagagctgaaagcagaagacatgatcattagccataagaaactcaaa ggaagaagaaataattagggaaagaagtctatttgatgaatatgtgtgtgtaaggta tgttctgctttcttggttcaaaaatgaagcgggcgttgtctagctcttaggtgaagg gagtctctgctttggaagaacggcacaggtaggacagaagtatcatccctaccccta actgatctgttattaaagctacaaattcttcacaccgtc  7 Mouse HFE2- ggctctctgacctgagtgagactgcagccattccggggcaatcatggagaaagagat NM_027126.4 gggggaccccctggctggagcagaccaacagaataggcaactatggctcgagaaccc agtatcagagtaatgcttgacctcgggaaacatcacagaagtacccagagaaattca ctaggtaggaggctcatcatctgggaagaaccggtgcctggggggacctggctggat aggtatgggccagtcccctagtccccggtccccccacggcagccctccaactccaag caccctcactctcctgctgctcctctgtggacaggctcactcccagtgcaagatcct ccgctgcaatgccgagtatgtctcgtccactctgagtcttcggggaggtggctcacc ggacacgccgcgtggaggcggccgtggtgggctggcctcaggtggcttgtgtcgcgc cctgcgctcctacgctctctgcacgcggcgcacggcccgcacctgccgcggggacct tgctttccactctgcggtgcatggcatagaggacctgatgatccagcacaactgctc acgccagggtcccacggccccgcccccggcccggggccccgccctgcccggggccgg gccagcgcccctgaccccagatccctgtgactatgaggcccggttttccaggctgca cggtcgagccccgggcttcttgcattgcgcatcctttggagatccccatgtgcgcag tttccacaaccaatttcacacatgccgtgtccaaggagcttggcccttgctagataa cgacttcctctttgtccaggccaccagctccccggtttcgtcgggagccaccgctac caccatccggaagatcactatcatatttaaaaacatgcaggaatgcattgaccagaa agtctaccaggctgaggtggacaatcttcctgcagcctttgaagatggttctatcaa tgggggcgaccgacctgggggctcgagtttgtccattcaaactgctaaccttgggag tcacgtggagattcgagctgcctacattggaacaactatcatcattcgacagacagc tgggcagctctccttctccatcagggtagcagaggatgtggcgcgggccttctccgc agagcaggacctacagctgtgtgttgggggatgccctccgagccagcgactctctcg ctcagagcgcaaccgccgtggggctatagccatagatactgccagaaggctgtgtaa ggaagggcttccggttgaagatgcctacttccaatcctgcgtctttgatgtttcagt ctccggtgaccccaactttactgtggcagctcagacagctctggacgatgcccgaat cttcttgacggatttagagaacttacatctctttccctcagatgcggggcctcccct ctctcctgccatctgcctagtcccgcttctttcggccctctttgttctgtggctttg cttcagtaagtaggccagcaacccatgactggtttggaaacgatttgaggatagagg ttggtgtgagaaaccacaaagatgtgccaaaggaaacagcggggacaggagacaaca cttacccaatcagatgaggttgcagtccagggctgaaatgaccctagaataaagatt ctgggccagggttttgcactccagaccttggtgtgggctattcaccatggatttccc agttagtgatttcccacttgtaatgaaattccactctccatacacctataccactcc ctacaagcctagagattgtgagagtgctaatgaccagtgaaacattaaaggactgag atatcgtaaaggcaaaaacatgattctctttgagaaagtcaaaagaggagaagctaa ttaggaaaagcttttggttcagaaacgaagtgggcattgtctggcagaggaagtcag cttttggagactggcaccaactcagaaacgggcatttccatcccttcctaatctgtt attaaagcgattagttctccatcctg  8 Rat HFE2- cggggacagacatggagaaggagatggaggaccccctggctggagcagaccaacaga NM_001012080 ataggcaactatggctggagaaccgggtatcagagtaatgcttgacctcgggaaaca .1 ccaaatttcttcttccgatcgcagaagtagtactcggcgaaattcactaggtaggag gctcctcatctgggaagaaccggtgcctggggggacctggctggataggtatggggg atcgaggccggtcccctagtctccggtccccccatggcagtcctccaactctaagca ccctcactctcctgctgctcctctgtggacaggctcactcccagtgcaagatcctcc gctgcaatgccgagtacgtctcgtccactctgagccttcggggagggggctcaccgg acacgccacatggaggcggccgtggtgggccggcctcaggtggcttgtgtcgcgccc tgcgctcctacgctctctgcacgcggcgcaccgcccgcacctgccgcggggacctcg ctttccactccgcggtgcatggcatagaggacctgatgatccagcacaactgctcac gccagggtcccacggcctcgcccccggcccggggtcctgccctgcccggggccggcc cagcgcccctgaccccagatccctgtgactatgaagcccggttttccaggctgcacg gtcgaaccccgggtttcttgcattgtgcttcctttggagacccccatgtgcgcagct tccacaatcactttcacacatgccgcgtccaaggagcttggcccctactagataacg acttcctctttgtccaagccaccagctccccggtagcatcgggagccaacgctacca ccatccggaagatcactatcatatttaaaaacatgcaggaatgcattgaccagaaag tctaccaggctgaggtagacaatcttcctgcagcctttgaagatggttctgtcaatg ggggcgaccgacctgggggctcgagtttgtccattcaaactgctaaccttgggagcc acgtggagattcgagctgcctacattggaacaactataatcgttcgtcagacagctg gacagctctccttctccatcagggtagcggaggatgtggcacgggccttctctgctg agcaggatctacagctgtgtgttgggggatgccctccgagccagcgactctctcgct cagagcgcaatcgccgtggggcgatagccatagatactgccagaaggttgtgtaagg aagggcttccggttgaagatgcctacttccaatcctgcgtctttgatgtttcagtct ccggtgaccccaactttactgtggcagctcagtcagctctggacgatgcccgagtct tcttgaccgatttggagaacttgcaccttttcccagtagatgcggggcctcccctct ctccagccacctgcctagtccggcttctttcggtcctctttgttctgtggttttgca ttcagtaagtaggccagcaacccgtgactagtttggaaacggtttgaggagagaggt tgatgtgagaaaacacaaagatgtgccaaaggaaacagtggggacaggagacaacga ccttactcaatcacacgaggttgcagtccagggctgaaatgaccctagaataaagat tctgagacagggttttgcactccagaccttggtatgggctccccatgaatttcccca ttagtgatttcccacttgtagtgaaattctactctctgtacacctgatatcactcct gcaaggctagagattgtgagagcgctaagggccagcaaaacattaaagggctgagat atcttaaaggcagaaactagaaaaggggaaaccatgattatctataagaaaatcaaa agaggggtttgggaatttagctcagtggtagagcacttgcctagcaagcgcaaggcc ctgggttcggtccccagctcctaaaaaagaaaaaaaaaatcaaaagagaaaaaacta attaaggcaagctttttggttcagaaatgaagtgggcattgtctggcagaggaagtc agcttttggagactggcaccaacatctccacccttcctactctgttattaaagtgac gaattccccaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagg  9 Human TFR2- cgctgggggacagcctgcaggcttcaggaggggacacaagcatggagcggctttggg NM_003227.3 gtctattccagagagcgcaacaactgtccccaagatcctctcagaccgtctaccagc gtgtggaaggcccccggaaagggcacctggaggaggaagaggaagacggggaggagg gggcggagacattggcccacttctgccccatggagctgaggggccctgagcccctgg gctctagacccaggcagccaaacctcattccctgggcggcagcaggacggagggctg ccccctacctggtcctgacggccctgctgatcttcactggggccttcctactgggct acgtcgccttccgagggtcctgccaggcgtgcggagactctgtgttggtggtcagtg aggatgtcaactatgagcctgacctggatttccaccagggcagactctactggagcg acctccaggccatgttcctgcagttcctgggggaggggcgcctggaggacaccatca ggcaaaccagccttcgggaacgggtggcaggctcggccgggatggccgctctgactc aggacattcgcgcggcgctctcccgccagaagctggaccacgtgtggaccgacacgc actacgtggggctgcaattcccggatccggctcaccccaacaccctgcactgggtcg atgaggccgggaaggtcggagagcagctgccgctggaggaccctgacgtctaccgcc cctacagcgccatcggcaacgtcacgggagagctggtgtacgcccactacgggcggc ccgaagacctgcaggacctgcgggccaggggcgtggatccagtgggccgcctgctgc tggtgcgcgtgggggtgatcagcttcgcccagaaggtgaccaatgctcaggacttcg gggctcaaggagtgctcatatacccagagccagcggacttctcccaggacccaccca agccaagcctgtccagccagcaggcagtgtatggacatgtgcacctgggaactggag acccctacacacctggcttcccttccttcaatcaaacccagttccctccagttgcat catcaggccttcccagcatcccagcccagcccatcagtgcagacattgcctcccgcc tgctgaggaagctcaaaggccctgtggccccccaagaatggcaggggagcctcctag gctccccttatcacctgggccccgggccacgactgcggctagtggtcaacaatcaca ggacctccacccccatcaacaacatcttcggctgcatcgaaggccgctcagagccag atcactacgttgtcatcggggcccagagggatgcatggggcccaggagcagctaaat ccgctgtggggacggctatactcctggagctggtgcggaccttttcctccatggtga gcaacggcttccggccccgcagaagtctcctcttcatcagctgggacggtggtgact ttggaagcgtgggctccacggagtggctagagggctacctcagcgtgctgcacctca aagccgtagtgtacgtgagcctggacaacgcagtgctgggggatgacaagtttcatg ccaagaccagcccccttctgacaagtctcattgagagtgtcctgaagcaggtggatt ctcccaaccacagtgggcagactctctatgaacaggtggtgttcaccaatcccagct gggatgctgaggtgatccggcccctacccatggacagcagtgcctattccttcacgg cctttgtgggagtccctgccgccgagttctcctttatggaggacgaccaggcctacc cattcccgcacacaaaggaggacacttatgagaacctgcataaggcgctgcaaggcc gcctgcccgccgtggcccaggccgtggcccagctcgcagggcagctcctcatccggc tcagccacgatcgcctgctgcccctcgacttcggccgctacggggacgtcgtcctca ggcacatcgggaacctcaacgagttctctggggacctcaaggcccgcgggctgaccc tgcagtgggtgtactcggcgcggggggactacatccgggcggcggaaaagctgcggc aggagacctacagctcggaggagagagacgagcgactgacacgcatgtacaacgtgc gcataatgcgggtggagttctacttcctttcccagtacgtgtcgccagccgactccc cgctccgccacatcttcacgggccgtggagaccacacgctgggcgccctgctggacc acctgcggctgctgcgctccaacagctccgggacccccggggccacctcctccactg gcttccaggagagccgtttccggcgtcagccagccctgctcacctggacgctgcaag gggcagccaatgcgcttagcggggatgtctggaacattgataacaacttctgaggcc ctggggatcctcacatccccgtcccccagtcaagagctcctctgctcctcgcttgaa tgattcagggtcagggaggtggctcagagtccacctctcattgctgatcaatttctc attacccctacacatctctccacggagcccagaccccagcacagatatccacacacc ccagccctgcagtgtagctgaccctaatgtgacggtcatactgtcggttaatcagag agtagcatcccttcaatcacagccccttcccctttctggggtcctccatacctagag accactctgggaggtttgctaggccctgggacctggccagctctgttagcgggagag atcgctggcaccatagccttatggccaacaggtggtctgtggtgaaaggggcgtgga gtttcaatatcaataaaccacctgatatcaataagccaaaa 10 Human TFR2- ccctgcccctggcgaccccacgtctctggcatccctccctcttccctccctctcctc NM_001206855 cgggcgcccagaaaagtccccacctctccccgcttaggcaaaccagccttcgggaac .1 gggtggcaggctcggccgggatggccgctctgactcaggacattcgcgcggcgctct cccgccagaagctggaccacgtgtggaccgacacgcactacgtggggctgcaattcc cggatccggctcaccccaacaccctgcactgggtcgatgaggccgggaaggtcggag agcagctgccgctggaggaccctgacgtctactgcccctacagcgccatcggcaacg tcacgggagagctggtgtacgcccactacgggcggcccgaagacctgcaggacctgc gggccaggggcgtggatccagtgggccgcctgctgctggtgcgcgtgggggtgatca gcttcgcccagaaggtgaccaatgctcaggacttcggggctcaaggagtgctcatat acccagagccagcggacttctcccaggacccacccaagccaagcctgtccagccagc aggcagtgtatggacatgtgcacctgggaactggagacccctacacacctggcttcc cttccttcaatcaaacccagttccctccagttgcatcatcaggccttcccagcatcc cagcccagcccatcagtgcagacattgcctcccgcctgctgaggaagctcaaaggcc ctgtggccccccaagaatggcaggggagcctcctaggctccccttatcacctgggcc ccgggccacgactgcggctagtggtcaacaatcacaggacctccacccccatcaaca acatcttcggctgcatcgaaggccgctcagagccagatcactacgttgtcatcgggg cccagagggatgcatggggcccaggagcagctaaatccgctgtggggacggctatac tcctggagctggtgcggaccttttcctccatggtgagcaacggcttccggccccgca gaagtctcctcttcatcagctgggacggtggtgactttggaagcgtgggctccacgg agtggctagaaggctacctcagcgtgctgcacctcaaagccgtagtgtacgtgagcc tggacaacgcagtgctgggggatgacaagtttcatgccaagaccagcccccttctga caagtctcattgagagtgtcctgaagcaggtggattctcccaaccacagtgggcaga ctctctatgaacaggtggtgttcaccaatcccagctgggatgctgaggtgatccggc ccctacccatggacagcagtgcctattccttcacggcctttgtgggagtccctgccg tcgagttctcctttatggaggacgaccaggcctacccattcctgcacacaaaggagg acacttatgagaacctgcataaggtgctgcaaggccgcctgcccgccgtggcccagg ccgtggcccagctcgcagggcagctcctcatccggctcagccacgatcgcctgctgc ccctcgacttcggccgctacggggacgtcgtcctcaggcacatcgggaacctcaacg agttctctggggacctcaaggcccgcgggctgaccctgcagtgggtgtactcggcgc ggggggactacatccgggcggcggaaaagctgcggcaggagatctacagctcggagg agagagacgagcgactgacacgcatgtacaacgtgcgcataatgcgggtggagttct acttcctttcccagtacgtgtcgccagccgactccccgttccgccacatcttcatgg gccgtggagaccacacgctgggcgccctgctggaccacctgcggctgctgcgctcca acagctccgggacccccggggccacctcctccactggcttccaggagagccgtttcc ggcgtcagctagccctgctcacctggacgctgcaaggggcagccaatgcgcttagcg gggatgtctggaacattgataacaacctctgaggccctggggatcctcacatccccg tcccccagtcaagagctcctctgctcctcgcttgaatgattcagggtcagggaggtg gctcagagtccacctctcattgctgatcaatttctcattacccctacacatctctcc acggagcccagaccccagcacagatatccacacaccccagccctgcagtgtagctga ccctaatgtgacggtcatactgtcggttaatcagagagtagcatcccttcaatcaca gccccttcccctttctggggtcctccatacctagagaccactctgggaggtttgcta ggccctgggacctggccagctctgttagtgggagagatcgctggcaccatagcccta tggccaacaggtggtctgtggtgaaaggggcgtggagtttcaatatcaataaaccac ctgatatcaataagccaaaa 11 Rhesus TFR2- accccaggacctgcgctcagggagcaggcaggtgtggggctgtggagagattggcag XM_001113151 gggagagcacagccgcttgtgctctggcctggactcaggggccacgtctggaaggtt .2 ggaccgaggccaggactgtgcccccacccttgggggtggtaaggagcagccttggct caggctttctgccagggctgataaggagccctcctggggctcccacaaacggtttat cggtttatcactggggacagcctgcaggcttcaggagggggcacaagcatggagcag ctttggggtctactccagagagcgcaacaactgtccccaagatcctctcagaccgtc taccagcgtgtggaaggcccccagaaagggcacctggaggaggaagaggaagacggg gaggagacactggcccacttctgccccatggagctgaagggccctgagcccctgggc tctagacccaggcagccaaacctcattccctgggcagcagcaggacggagggctgcc ccctacctggtcctgactgctctactgatcttcactggggccttccttctgggctac gtcgccttccgagggtcctgccagacatgcggagactccgtgttggtggtcagtgag gacgtcaactatgagcctgacctggatttccaccggggcacactgtactggagcgac ctccaggccatgttcctgcagttcccgggggaggggcgcctggaggacaccatcagg caaaccagccctcgggaacgggtggcaggctcggccgggatggccgctctgactcag gatatccgcgcggcgctctctcgccagaaactggaccacgtgtggaccgacacgcac tacgtggggctgcaattcccggacccggctcaccccaacaccctgcactgggtcgat gaggccgggaaggtcggagagcagctgccgctagaggaccctgacgtctactgcccc tacagcgccatcggcaacgtcacgggagagctggtgtacgcccactacgggcggccc gaagacctgcaggacctgcgggccaggggcgtggacccagcgggccgcctgctgcta gtgcgcgtgggggtgatcagcttcgcccagaaggtgaccaatgctcaggactttggg gctcaaggagtgctcatatacccagagccagcggacttctcccaggacccacacaag ccaagcctgtccagccagcaggctgtgtatggacatgtgcacctgggaactggagac ccctacacgcctggcttcccttccttcaatcaaacccagttccctccagttgcatca tcgggccttcccagcatcccagcccagcccatcactgcagacattgcctcccgcctg ctgaggaagctcaaaggccctgcggccccccaggaatggcaggggagcctcctaggc tccccttatcacctgggccccgggccacgactgcggctagcggtcaacaaccacagg acctccacccccatcaacaacatctttggctgcatcgaaggccgctcagagccagat cactatgttgtcatcggggcccagagggatgcgtggggcccaggagcagctaaatcc gctgtggggacagctatactcctggagctggtgcggaccttttcctccatggtgagc aacggcttccggccccgcagaagtctcctcttcatcagctgggatggcggtgacttt gggagcgtgggctccacagagtggctagagggctacctcagtgtgctgcacctcaaa gctgtagtgtacgtgagcctggacaacgcagtgccgggggatgacaagtttcatgcc aagaccagcccccttctgacaagtctcattgagagtgtcctgaaacaggcaagagca ccccaggaatggctgaccctgcagtgggtgtactccgcgcggggggactacatccgg gcggcggagaagctgcggcaggagatctacagctcggaggagagagacgagcgactg acacgcatgtacaacgtgcgcataatgcgggtggagttctacttcctttcccagtac gtgtcgccggccgactccccgttccgccacatcttcatgggccgcggagaccacacg ctgggcgccctgctggaccacctgcggctgctgcgctccaacagctccgggaccccc ggggccacctcctccgccgtcttccaggagagtcgcttccggcgtcagctagccctg ctcacctggacgctgcaaggggcagccaatgcgcttagcggggacgtctggaacatt gataacaacttctgagaccctggggatcctcagatccccctgtccccttgtcgagag ctcctctgctcctcgcttcaatgattcagggtcagggaggtggctcagagtccacct ctcattgctgatcgacttctcattacccctacacgtctctccacggagcccagactg cagcacagatatccacacaccccagccctgcagtgtagctgactctaatgtgatggt catactgtcggttaatcagagagcagtatcccttcaatcacaaccccttcccctttc tggggtcctccatacctagagactaggccttgggacctggccagctctcttagcggg agagatcgctggcaccatagccttatggccaacaggtggtctgtggtgaaaggggca tggagtttcaatgtc 12 Mouse TRF2- gagcatggtccaagaaacccagagacctgttgctgagctgaacttggctgctgtgtc NM_015799.3 ttcccactcaggactcggctttgacagctgcaggtcctggtgtcttcgtcgcggctt ggatttcaaactggaggagttcaggagggggcacaagcatggagcaacgttggggtc tacttcggagagtgcaacagtggtccccaagaccctctcagaccatctacagacgcg tggaaggccctcagctggagcacctggaggaggaagacagggaggaaggggcggagc ttcctgcccagttctgccccatggaactcaaaggccctgagcacttaggctcctgtc ccgggaggtcaattcccataccctgggctgcagcaggtcgaaaggctgccccctatc tggtcctgatcaccctgctaatcttcactggggccttcctcctaggctacgtggcct ttcgagggtcctgccaggcgtgtggggactccgtgttggtggtcgatgaagatgtca accctgaggactccggccggaccacgttgtactggagcgacctccaggccatgtttc tccggttccttggggaggggcgcatggaagacaccatcaggctgaccagcctccggg aacgcgtggctggctcagccagaatggccaccctggtccaagatatcctcgataagc tctcgcgccagaagctggaccacgtgtggactgacacgcactacgtgggacttcagt tcccagatccggctcacgctaacaccctgcactgggtggatgcagacgggagcgtcc aggagcagctaccgctgaaggatccggaagtctactgcccctacagcgccaccggca acgccacgggcaagccggtgtacgcccactacgggcggtcggaggacctacaggacc taaaagccaagggcgtggagctggccggcagcctcctgctagcgcgagttggaatta ctagcttcgcccagaaggtagccgttgcccaggactttggggctcaaggagtgctga tataccctgacccatcagacttctcccaggatccccacaagccaggcctgtctagcc accaggctgtgtacggacatgcgcacctgggaactggagacccttacacacctggct tcccgtccttcaatcaaacccagttccctccagtagaatcatcaggccttcccagca tccccgcccagcccatcagtgctgacattgctgaccaattgctcaggaaactcacag gccccgtggctccccaggagtggaaaggtcacctctcaggctctccttatcggctgg gacctgggcccgacttacgccttgtggtcaacaaccacagagtctctacccccatca gtaacatctttgcgtgcatcgagggctttgcagagccagatcactatgttgtcattg gggcccagagggatgcatggggcccaggagcagccaagtctgcagtggggactgcca tcctgctggagctggttcggaccttctcttccatggtcagcaatgggttcagacctc gaagaagtcttttgttcatcagctgggacggaggtgactttggcagcgtgggagcca cagagtggttggagggctacctcagcgtgctacacctcaaagctgttgtgtacgtga gcctggacaactccgtgttgggagatggcaaattccatgctaagaccagcccccttc tcgtcagcctcattgagaatatcttgaagcaggtggactcccctaaccatagtggac agaccctctatgaacaagtggcactcacccaccccagctgggatgctgaagtgattc agcccctgcccatggacagcagtgcatattccttcacagcctttgcgggggtcccag ccgtggagtcctccttcatggaggacgatcgggtgtacccattcctgcacacgaagg aggacacatacgagaatctgcacaagatgctgcgaggtcgcccgcccgccgtggtcc aggcagtggctcagctcgcgggccagctcctcatccgactgagccacgatcacctac tgccgctagacttcggccgctatggagacgcggttctcaggcacatcggcaacctca atgagttctctggggacctcaaggagcgcgggctgaccctgcagtgggtgtactctg caaggggggactacatccgtgcggcggaaaagctgcggaaggagatttacagctcgg agcggaacgatgagcgtctgatgcgcatgtacaacgtgcgcatcatgagggtggagt tctacttcctgtcccagtatgtgtcgccagccgactccccattccgccacattttcc taggccaaggcgaccacactttgggtgccctggtagaccacctgcggatgctgcgcg ccgatggctcaggagccgcctcttcccggttgacagcaggtctgggcttccaggaga gtcgcttccggcgccagctggcgctgctcacctggacactgcagggggcagccaacg ctctcagtggcgacgtttggaacattgacaataacttttgaagccaaaagccctcca tgggccccacgtgattctcctttctccctctttgagtggtgcaggcaaaggaggcgc ctgagattgtaacctattcttaacacccttggtcctgcaatgctggtgcgccatatt ttctcagtgtggttgtcatgccgttgcttacccagaaagcggttttcttcccatcac aggcccttctgtcttcaggagcaaagttccccatatctagagactatctagatgctg ggatctgatcagctctcttagagagtgagatggacagcgtcattattttatgacaca tgagctacggtatgtgagcagcccaaggggattagatgtcaataaaccaattgtaac ccctgttgtccatacgcaa 13 Rat TFR2- aaatccagagacctgttgctgagttgaacttggctgctgtgtcttcccactcaggac NM_001105916 tcggctttgacagacacgaggcagggactggggtgagcccctacctctcagatcttt .1 ctggacctggctgcgggtcctgggatcttcagcgcggcttggatttcaaactggagg ggttcaggagggggcacaagcatggaacaacgttggggtctacttcggaaagtgcaa cagtggtccccaagaccctctcagaccatctacagacgtgtggaaggccctcaactg gagaacctagaggaggaagatagggaggaaggggaggagcttcctgcccagttctgc cccatggaactcaaaggccctgagcgcttaggctcctgtcctgggaggtccattccc ataccctgggctgcagcaggtcgaaaggctgctccctatctggtcctgaccaccctg ctaatcttcactggggccttcctcctgggctacgtggcctttcgagggtcctgccag gcatgtggggactctgtgttggtggttggtgaagatgtcaactctgaggactccagc cggggcacgttgtactggagtgacctccaggacatgtttctccggttccttggggag ggacgcatggaggacaccatcaggctgaccagcctccgggaacgcgtggccggctca gccagaatggccaccctggtccaagacatcctcgataagctctcgcgccagaagctg gaccacgtgtggactgacacgcactatgtgggacttcagttcccggacccggctcac cctaacaccctgcactgggtgggtgcagacgggagcgtccaagagcagctaccgctg gaggatccggaagtctactgtccctacagcgccacgggcaacgccacgggcaagctt gtgtacgcccactacgggcggcgggaggacctgcaggacctgaaagccaaggacgtg gagctggccggcagcctcctgctagtgcgcgctgggattacaagcttcgcccagaag gtagccattgcccaggactttggggcccacggagtgctgatataccctgacccagcg gacttctcccaagacccccacaagccaggcctgcctagtgacagggctgcgtatgga catgtgcacctgggaactggggacccttacacgcctggcttcccgtccttcaatcaa acccagttccctccagtagaatcatcggggcttcccaacatccctgcccagcccatc agtgccgacgttgctgatcgcttgctcaggaaactcacaggtcccgtggctcctcag gaatggaagggtcgcctctcagactctccgtatcgcctgggacctgggccaggctta cgccttgtggtcaacaaccacagaacctctactcccatcagtaacatctttgcgtgc atcgagggcttcgcagagccagatcactatgtcgttatcggggcccagagggatgcc tggggcccaggagcagccaagtctgcagtggggactgccatcctcctggagctggtt cggaccttttcctccatggtcagcagtggctttagacctcgaagaagtcttttgttc atcagctgggacggaggtgactttggcagcgtgggagccacggagtggttggagggc tacctcagcgtgctacacctcaaagctgtcgtgtatgtgagcctggacaactccgtg ttgggagacggcaaattccatgctaagaccagcccccttctcgtcagcctcattgag aatatcctgaagcaggtggattcccctaaccacagtggacagacactctacgatcaa gtggcattcacccacccaagctgggatgctgaagcgatccagcccctgcccatggac agcagcgcatattccttcacagcttttgcgggcgtcccagctgtggagttctccttc atggaggacgatagggtgtacccattcctgcacacgaaggaggacacgtatgagaat ctgcacaagatgctgcgaggtcgcctgcccgccgtggtcctagcagtggctcagctc gctggtcagctcctcatccgactgagccacgatcacctactgccgctggacttcggc cgctacggagacgtggtcctcaggcacatcggcaaccttaatgagttctctggggac ctcaaggcgcgcgggctgaccctgcagtgggtgtactctgcaaggggggactatatc cgggcggcggagaagctgcggaaggagatttacagctcggagcagagcgatgagcgt ctgatgcgcatgtacaacgtgcgcatcatgagggtggagttctacttcctgtcccag tacgtgtcgccggccgactccccattccgccacattttcctaggccaaggcgaccac actttgggtgccctggtggaacacctacggatgctgcgctccgatggctcaggagct gcctcttctgggttgagcccaggtctgggcttccaggagagtcgcttccggcgacag ctggcgctgctcacgtggacgctacagggggcagccaacgcactcagtggcgacgtt tggaacatcgacaataacttttgaggccagaagtcctccatgggccccacgtgattc tcctttctccctatttgagtggtgcaggcaacggaggtgcctgagagcaacctatcc tcattaacaaccttggtcctgcaacgccagtgagacatattttctcagtgtgactgt tataccactgtttatccagaaagcggttttcttcccatcactggcctctctgccttc aggagcatagttccccatatctagaaaccatctagacactgggatccagctctctta gcgggtgagatggatagcgtcatttccttatgacacacaagtggtatgtgggtggcc caagggggattagatgtcaataaaccatttacctggtaacctctgttgtccataagc 14 Human HFE- ctaaagttctgaaagacctgttgcttttcaccaggaagttttactgggcatctcctg NM_139006.2 agcctaggcaatagctgtagggtgacttctggagccatccccgtttccccgcccccc aaaagaagcggagatttaacggggacgtgcggccagagctggggaaatgggcccgcg agccaggccggcgcttctcctcctgatgcttttgcagaccgcggtcctgcaggggcg cttgccgcgttcacactctctgcactacctcttcatgggtgcctcagagcaggacct tggtctttccttgtttgaagctttgggctacgtggatgaccagctgttcgtgttcta tgatcatgagagtcgccgtgtggagccccgaactccatgggtttccagtagaatttc aagccagatgtggctgcagctgagtcagagtctgaaagggtgggatcacacgttcac tgttgacttctggactattatggaaaatcacaaccacagcaaggagtcccacaccct gcaggtcatcctgggctgtgaaatgcaagaagacaacagtaccgagggctactggaa gtacgggtatgatgggcaggaccaccttgaattctgccctgacacactggattggag agcagcagaacccagggcctggcccaccaagctggagtgggaaaggcacaagattcg ggccaggcagaacagggcctacctggagagggactgccctgcacagctgcagcagtt gctggagctggggagaggtgttttggaccaacaagtgaccactctacggtgtcgggc cttgaactactacccccagaacatcaccatgaagtggctgaaggataagcagccaat ggatgccaaggagttcgaacctaaagacgtattgcccaatggggatgggacctacca gggctggataaccttggccgtaccccctggggaagagcagagatatacgtgccaggt ggagcacccaggcctggatcagcccctcattgtgatctgggagccctcaccgtctgg caccctagtcattggagtcatcagtggaattgctgtttttgtcgtcatcttgttcat tggaatcttgttcataatattaaggaagaggcagggttcaagaggagccatggggca ctacgtcttagctgaacgtgagtgacacgcagcctgcagactcactgtgggaaggag acaaaactagagactcaaagagggagtgcatttatgagctcttcatgtttcaggaga gagttgaacctaaacatagaaattgcctgacgaactccttgattttagccttctctg ttcatctcctcaaaaagatttccccatttaggtttctgagttcctgcatgccggtga tccctagctgtgacctctcccctggaactgtctctcatgaacctcaagctgcaccta gaggcttccttcatttcctccgtcacctcagagacatacacctatgtcatttcattt cctatttttggaagaggactccttaaatttgggggacttacatgattcattttaaca tctgagaaaagctttgaaccctgggacgtggctagtcataaccttaccagattttta cacatgtatctatgcattttctggacccgttcaacttttcctttgaatcctctctct gtgttacccagtaactcatctgtcaccaagccttggggattcttccatctgattgtg atgtgagttgcacagctatgaaggctgtacactgcacgaatggaagaggcacctgtc ccagaaaaagcatcatggctatctgtgggtagtatgatgggtgtttttagcaggtag gaggcaaatatcttgaaaggggttgtgaagaggtgttttttctaattggcatgaagg tgtcatacagatttgcaaagtttaatggtgccttcatttgggatgctactctagtat tccagacctgaagaatcacaataattttctacctggtctctccttgttctgataatg aaaattatgataaggatgataaaagcacttacttcgtgtccgactcttctgagcacc tacttacatgcattactgcatgcacttcttacaacaattctacgagataggtactat tatccccatttcttttttaaatgaagaaagtgaagtaggccgggcacggtggctcac gcctgtaatcccag 15 Rhesus HFE- ttttactgggcatctcctgagcctaggcaatagctgtagggtgacttctggagccat XM_001085598 cgccgtttccccgccccaccaaagaagcggagacttaaaggggacgtgcagtcagag .2 ctggggaaatgggcccgcgagccaggccggcgcttctcctcctgatgcttttgcaga ccgcggtcctgcaggggcgcttgctgcgttcacactctctgcactacctcttcatgg gttcctcagagcaggaccttggtctttccctgtttgaagctttgggctatgtggacg accagctgttcgtgttccatgatcacgagagtcgccgtgtggagccccgaactccat gggtttccggtagaacgtcaagccagatgtggctgcagctgagtcagagtctgaaag ggtgggatcacatgttcactgttgacttctggactattatggaaaatcacaaccaca gcaaggagtcccacaccctgcaggtcatcctgggctgcaaaatgcaagaggacaaca gtaccgagggcttctggaagtacgggcacgatgggcaggaccaccttgaattctgcc ctgacacactggattggagagcagcagaacccagggcctggcccaccaagctggagt gggaaaggcacaaaattcgggccaggcagaacagggcctacctcgagagggactgcc ctgtgcagctgcagcagttgctggagctggggagaggtgttttcgaccggccagtga ccactctacggtgtcgggccctgaactactacccccagaacatcaccatgaagtggc tgaaggataggcagtcaatggatgccaaggaggtcgaacctaaagacgtattgccca atggggatgggacctaccagggctggataaccttgactgtacccccaggggaagaac agagatatacttgccaggtggagcacccaggcctggatcagcccctccttgctttct gggagccctcaccatctagaactctagtcattggagtcatcagtggaattgctgttt ttgtcatcatcttgttcattggaattttgttcataatattaaggaagaggcagactt caagaggagtcatggggcactacgtcttagctgaacgtgagtgacacg 16 Mouse HFE- ctgagaggtctggaacctcagcaatggctacagggtgacttcttggatcctccacgt NM_010424.4 ttccagatcctagtgaagaccggtggacccagctgaggacatgagcctatcagctgg gctccctgtgcggccgctgctgctgctgctgctactgctgtggtccgtggccccgca ggcactgccaccgcgttcacattctccaagatacctcttcatgggtgcctcagagcc agacctcgggctgcctttgtttgaggctaggggctatgtggatgaccagctctttgt gtcctacaatcatgagagtcgccgtgctgagcccagggccccgtggatcttggagca aacctcaagccagctgtggctgcatctgagtcagagcctgaaagggtgggactacat gttcatagtagacttctggaccatcatgggcaactataaccacagtaaggtcacgaa gttgggagtggtgtccgagtcccacatcctgcaggtggtcctaggctgtgaggtgca tgaagacaacagtaccagcggcctctggagatatggttatgacgggcaagatcacct ggaattctgccccaagacactaaactggagcgcagccgagccaggggcctgggccac caaggtggaatgggacgagcacaagatccgtgccaaacagaacagggactacctgga gaaggactgccccgagcagctgaaacggctcctggagctggggagaggcgttctggg acagcaagtgcctactttggtgaaagtgactcgccactgggcctctacggggacctc tctaaggtgtcaggctctggacttcttcccccagaacatcactatgaggtggttgaa ggacaaccaaccactggatgccaaagatgtcaaccccgagaaggtgctacctaacgg ggatgagacctatcaaggctggctgacattggccgtggcccctggggacgagacaag gttcacctgtcaagtggagcacccaggcctggaccagcctctcactgcctcttggga gcccttgcaatctcaggccatgattatcggaatcatcagtggagtcaccgtctgtgc catctccttggttggaattctgttcctaatcttaaggaaaaggaaggcttcaggagg aaccatgggtggctatgtcttaacagactgtgagtgatctgcagcctgctgaaccac ggaagagagaaaactcagccaaagacttggagggggcacacttgctccactgtagga cacagttggacctaacacacagaaactgcctgagaactgtgctcttagctttctctg ttcactttcttaaggtgttttctccagttaagttcagttcctgaatagtagtgattg caccagttgcaacctctccctccagaactggtctcatgattcttaggctgcttcttg gaagcatcctatgtttccttcatgcacctagactccatatgtctacgtaaagagccc ctctaagtttagtggatacatgattcgtttccacatctgaagaagttgtgaaccttc atccggggatgctcacacatacttgagccagaatttttcacctatatcctagaatcc aggacccactcaactatcctccatctgttatagagtgactcctctgtcaccatgccc tgacttctctgccattggagtgttatatatatggatcatcaataaagccatgaaggc tacacaactgtg 17 Rat HFE- tcagcaatggctacagggtgacttcttggatcctccacgtttccaggtcctagtgaa NM_001173435 aaccggtggacccagctggaggcatggaccgatcagctgggctccctgtgcggctgc .1 tattgctgctgctgttgttgctgctgtggtccgtggccccgcaggcgctgcggcccg tgcctacttcggtgaaagtgactcgccactgggcctctacagggacctccctaaggt gtcaggctctgaatttcttcccccagaacatcactatgaggtggttgaaggacagcc agcccctagatgccaaggatgtcaaccctgagaacgtgctgccaaatggggatggga cctatcagggctggctgaccttggctgtggcccctggagaagagacaaggttcagct gtcaagtggagcacccaggcctggatcagcctctcactgccacttgggagccctcac ggtctcaggacatgattattggaatcataagtgggatcaccatttgtgccatcttct ttgttggaattctgatcctagtcttaaggaaaaggaaggtttcaggaggaaccatgg gtgactatgtcttaacagagtgtgagtgacctgcagcatgcagaagcacagaagaga gaagactcagccaaagacttggaggggacacacttgctccattctagaacacagctg gacctaacacacagaaactgcctgaggactctgcccttagctttcctgtttgctttc ttaaggtgttttctccagttaagttcagttcctgaataatagtgactgccccagctg caacctctcccttcagaaccagtctcatgatctttaagctgctacttgcaggcatcc ttcgttttctgcatccacctagacttcgtatgtctacttaaaaagccccactaaatt tgggggacacatgattcatttccacatctgaagaagttatgaaccttcatcctggga tgcacacattcttgtgccagaatttttcatacatatcctaggacccattcaattgtc atttgagcccctctatccgttagtgactactctgacttctctgccattggagtgtta tggcaataaagctatgaacgtta 18 Mouse BMPR1a- ccgcgcgagacgacgactgtacggccgcgcgaggggcgaccgggcccgggccgctgc NM_009758.4 acgccgagggcggaggccgagccgggccccgccgccccgcggctgtccgtgcccgcc cgcgccgagcgccggaggatgagtttctcgggatcccgatttatgaaaatatgcatc gctttgatactgtctggaattccatgagatggaagcataggtcaaagctgttcggag aaattggaactacagttttatctagccacatctctgagaattctgaagaaagcagca ggtgaaagtcattgccaagtgattttgttctgtaaggaagcctccctcattcactta caccagtgagacagcaggaccagtcattcaaagggccgtgtacaggacgcgtgcgaa tcagacaatgactcagctatacacttacatcagattactgggagcctgtctgttcat catttctcatgttcaagggcagaatctagatagtatgctccatggcactggtatgaa atcagacttggaccagaagaagccagaaaatggagtgactttagcaccagaggatac cttgcctttcttaaagtgctattgctcaggacactgcccagatgatgctattaataa cacatgcataactaatggccattgctctgccatcatagaagaagatgatcagggaga aaccacattaacttctgggtgtatgaagtatgaaggctctgattttcaatgcaagga ttcaccgaaagcccagctacgcaggacaacagaatgtcgtcggaccaatttgtgcaa ccagtatttgcagcctacactgccccctgttgttataggtccgttctttgatggcag catccgatggctggttgtgctcatttccatggctgtctgtatagttgctatgatcat cttctccagctgcttttgctataagcattattgtaagagtatctcaagcaggggtcg ttacaaccgtgatttggaacaggatgaagcatttattccagtaggagaatcattgaa agacctgattgaccagtcccaaagctctgggagtggatctggattgcctttattggt tcagcgaactattgccaaacagattcagatggttcggcaggttggtaaaggccgcta tggagaagtatggatgggtaaatggcgtggtgaaaaagtggctgtcaaagtgttttt taccactgaagaagctagctggtttagagaaacagaaatctaccagacggtgttaat gcgtcatgaaaatatacttggttttatagctgcagacattaaaggcactggttcctg gactcagctgtatttgattactgattaccatgaaaatggatctctctatgactccct gaaatgtgccacactagacaccagagccctactcaagttagcttattctgctgcttg tggtctgtgccacctccacacagaaatttatggtacccaagggaagcctgcaattgc tcatcgagacctgaagagcaaaaacatccttattaagaaaaatggaagttgctgtat tgctgacctgggcctagctgttaaattcaacagtgatacaaatgaagttgacatacc cttgaataccagggtgggcaccaagcggtacatggctccagaagtgctggatgaaag cctgaataaaaaccatttccagccctacatcatggctgacatctatagctttggttt gatcatttgggaaatggctcgtcgtcgtattacaggaggaatcgtggaggaatatca attaccatattacaacatggtgcccagtgacccatcctatgaggacatgcgtgaggt tgtgtgtgtgaaacgcttgcggccaatcgtgtctaaccgctggaacagcgatgaatg tcttcgagcagttttgaagctaatgtcagaatgttgggcccataatccagcctccag actcacagctttgagaatcaagaagacacttgcaaaaatggttgaatcccaggatgt aaagatttgacaattaaacaattttgagggagaatttagactgcaagaacttcttca cccaaggaatgggtgggattagcatggaataggatgtcgacttggtttccagactcc ttcctctacatcttcacaggctgctaacagtaaaccttaccgtactctacagaatac aagattggaacttggaacttcaaacatgtcattctttatatatggacagctttgttt taaatgtggggtttttttgttttgctttttttgttttgttttggttttgatgctttt ttggtttttatgaactgcatcaagactccaatcctgataagaagtctctggtcaacc tctgggtactcactatcctgtccataaagtggtgctttctgtgaaagccttaagaaa attaatgagctcagcagagatggaaaaaggcatatttgccttctaccagagaaaaca tctgtctgtgttctgtctttgtaaacagcctatagattatgatctctttgggatact gcctggtttatgatggtgcaccatacctttgatatgcataccagaattctctgctgc cctagggcttagaagacaagaatgtaaaggttgcacaggaaggtatttgtggccagt ggtttaaatatgcaatatctagttgacaatcgccaatttcataaaagccatccacct tgtaactgtagtaacttctccactgactttatttttagcataatagttgtgaaggcc aaactccatgtaaagtgtccatagacttggactgttttcccccagtcaccattttgt tctccttttggtaattatttttgttataaaaagccacctatccagaattggagctct ctgtcttgaaccatactttgaaagaaacgcctcttccgtactgcatctgatcacaat gtgcatacctatgatcaaattctggagtctttgttctcggtacctcctaaaaaggaa agttgattcttgtgcaacatgcttttattttcagaacctgcacagctgtcattctag ccatgttttacctacacactcagttctacacaagacagcccatacactctgtctcac atctgatccttggtgggaagtgttttaaagtagaactatgtatgaatttcagaattc atgcattttaaaacttcactaagatattgtctcatatctttatgagaatgtcagctg acttttcaactaacagtaaatgtattttagatatctaaatcttttgaaatttggttt tacaatttctggtccctaattgtgaagacaagaggcagaagtacccagtcactaccc atatttacactgaacgttattaaataaaatgatgtgtattttattataaaataaata taggccttgttatctcaaaaaacagatctggttcaaacttattataccaatatcata ctatttaaatgttctaagtaaacaagccatgtgagcatcaagtggcattggctcttt ggatgaaacataaacttaaggtgattgtatcaacacatagagtgactgaaattaaat gggaggcaggtagagcatatgtccatctgtccacctacaggcatgactaaactacag ctcatattccacaaatttgagatttgtcttgcctggtttgtttagtgagtctcatct gatgtacctaaagcctgagagtactgaggtctgattttatatctttcccgaataaac taaatcttttttgtcacttatcatcttaatgatatacctaaggaataattctttggc atgtttcagttgtgcgtggcagccactgtaatgactcttctctaagaaaggctgtca ggagttaattataaggcaggcagtgagcgctctagtcactgccttcccacgctgcca tcactgcattcatgggaatcagtgacgttctcgaaatggcaaacgctgctgcttttc cttatttggaatcctaaaatcaaaagttgcattaaacttactgtgttctcttatccc tctcagccataaatgtaaaattcagtaagtaaaaatatttaaagagtgtatcagccc tttggccagtgagatagctcagtagataaaggcatttgctgccaagtcctcaacctc aattcagactctgggggacacatggcgaagggaggagccaactaccccatcattgtc ctctgacttccacacactccatggcttgcgcccctccccacagacacacaccatgta ctccacaaaagtagtttaaaggaaaaaaagaatagaacccactgtgtaatggaataa gtattatgtagttacttaacaacttgtaaaaatctggaaactacgatttggttcccc tttgaatctagagtttaaaaaacagatggctaaaatcagccatcatttaaataatta aaaataaaagccccaaaccccaaactgcctaaataaataccaagtaatccaggaagc cgtcatgtgtggtttgtatgaccagtagttctctggtacagagcatgttaagatttg ccccagcctgattctctgaggtctctgcattactgagtactgtcctgagtataaaat ctgaactgatttctctagaaatactgtaacaaaaaggtattttagtcagcatgttat gttaacccttccactgtctagaaacttgaataagcacataaagacaccttttgctgt catcatctgttgtcctggaatgtgccagttttaatttattcattctaatgatattca atttgcttttcttttttagatgtttttcttgtttagagtaaaaggacgaatttttca agaaccttgcatctctgatttggcctaaggtcaaattggatattgagtagtctattc cagggcagatttcctaagcaatacttgtcttttcagctatgtattgttttgaaatgt ttccatttcaacagaggtgtaagtcatgtgaaaagaaaggtggtgtagcccttgtgg taatgacacaagttgacttgcgtcagatgttaagcagggacagttctcccacctcct ggctgtaaggagtggaaactaggcaagcagtgtatcagtccacagaggacaggaagg gtcatcccataaagaaagcctgtgagtatggctttggcaaaaaattagacataatac tgtccttttaggttgtgctctgttctttcctttcagtggaattatttaagctcttta gtggcctttgtttttcccacttaaaaactaaaatgtagcatatattgtataaaatgg aaatattaatagcttagggaaactgtacataaggcattgacaggtttaaaaaaagca tttttattatgcagttgtaaaacaccaaaaatatagattcatcttgatatgtaacac taagtgtattttgtacagcatctgatttgaaaggtgccttatgaagtttaccattaa ttgctttgttctatatacagattatgtccaatgtatcatttttcagtaaataacctt attttagta 19 Rat BMPR1a- gaattcatgagatggaaacataggtcaaagctgtttggagaaattggaactacagtt NM_030849.1 ttatctagccacatctctgagaagtctgaagaaagcagcaggtgaaagtcattgtca agtgattttgttcttctgtaaggaaacctcgttcagtaaggccgtttacttcagtga aacagcaggaccagtaatcaaggtggcccggacaggacacgtgcgaattggacaatg actcagctatacacttacatcagattactgggagcctgtctgttcaccatttctcat gttcaagggcagaatctagatagtatgctccatggtactggtatgaaatcagacgtg gaccagaagaagccggaaaatggagtgacgttagcaccagaggacaccttacctttc ttaaaatgctattgctcaggacactgcccagatgacgctattaataacacatgcata actaatggccattgctttgccattatagaagaagatgatcagggagaaaccacgtta acttctgggtgtatgaagtatgaaggctctgattttcaatgcaaggattcaccaaaa gcccagctacgcaggacaatagaatgttgtcggaccaatttgtgcaaccaatatttg cagcctacactgccccctgtcgttataggcccattctttgatggcagcgtccgatgg ctggctgtgctcatctctatggctgtctgtattgtcgccatgatcgtcttctccagc tgcttctgttacaaacattactgtaagagtatctcaagcagaggtcgttacaaccgt gacttggaacaggatgaagcatttattccagtaggagaatcactgaaagacctgatt gaccagtcacaaagctctggtagtggatctggattacctttattggttcagcgaact attgccaaacagattcagatggttcggcaggttggtaaaggccggtatggagaagta tggatgggtaaatggcgtggtgaaaaagtggctgtcaaagtattttttaccactgaa gaagctagctggtttagagaaacagaaatctaccagacggtgttaatgcgtcatgaa aatatacttggttttatagctgcagacattaaaggcaccggttcctggactcagctg tatttgattactgattaccatgagaatgggtctctctatgacttcctgaaatgtgcc accctggacaccagagccctactcaagttagcttattctgctgcctgtggtctgtgc cacctccacacagaaatttatggcacgcaaggcaagcctgcaattgctcatcgagac ctgaagagcaaaaacatccttattaagaaaaatggtagttgctgtattgctgacctg ggcctagctgttaaattcaacagtgacacaaatgaagttgacatacccttgaacacc agggtgggcaccaggcggtacatggctccagaagtgctggacgagagcctgagtaaa aaccatttccagccctacatcatggctgacatctacagctttggtttgatcatttgg gagatggcccgtcgctgtattacaggaggaatcgtggaggaatatcaattaccatat tacaacatggtgcctagtgacccatcttatgaagacatgcgtgaggtcgtgtgtgtg aaacgcttgcggccaatcgtctctaaccgctggaacagtgatgaatgtcttcgagcc gttttgaagctgatgtcagaatgctgggcccataatccagcatccagactcacagct ttgagaatcaagaagacgctcgcaaagatggttgaatcccaggatgtaaagatttga caaacagttttgagaaagaatttagactgcaagaaattcacccgaggaagggtggag ttagcatggactaggatgtcggcttggtttccagactctctcctctacatcttcaca ggctgctaacagtaaactttcaggactctgcagaatgcagggttggagcttcagaca taggacttcagacatgctgttctttgcgtatggacagctttgttttaaatgtgggct tttgatgcctttttggtttttatgaattgcatcaagactccaatcctgataagaagt ctctggtcaaactctggttactcactatcctgtccataaagtggtgctttctgtgaa agccttaaggaaattagtgagctcagcagagatggagaaaggcatatttgccctcta cagagaaaatatctgtctgtgttctgtctctgtaaacagcctggactatgatctctt tgggatgctgcctggttgatgatggtgcatcatgcctctgatatgcataccagactt cctctgctgccatgggcttacaagacaagaatgtgaaggttgcacaggacggtattt gtggccagtggtttaaatatgcaatatctaatcgacattcgccaatctcataaaagc catctaccttgtaactgaagtaacttctctaccaactttatttttagcataatagtt gtaaaggccaaactatgtataaagtgtccatagactcgaactgttttcctccagtca ccattttgttttccttttggtaattatttttgttatataatccctcctatccagaat tggcgctcactgtcttgaaccatactttgaaagaaatgcctcttcctggagtctgcc ttactgcatctgatcaccatgtgcatacctctgatcaaattctggagtctttgttct cggtacctcttaaaaagggaaattgtgtatcatgtgtagtgtgcttttattttcaaa atcttcatagcctttattctagccatttttacctacatactcattctgtacaaaaca gctcactcggtctcacggctgatcctcagtggaaatgatttaaagtagagctgtgta cgaatttcagaattcatgtatttaaaaacttcacactaacactttactaagatattg tctcatatcttttatgaggatgtcagctgattttcaatgactataaatgtatcttag ctatctaaatcttttgaaatttggttttataatttctggtccctaacttgtgaagac aaagaggcagaagtacccagtctaccacatttacactgtacattattaaataaaaaa atgtatattttaaaaaaaaaaaaaaaaaaaaa 20 Human SMAD4- atgctcagtggcttctcgacaagttggcagcaacaacacggccctggtcgtcgtcgc NM_005359.5 cgctgcggtaacggagcggtttgggtggcggagcctgcgttcgcgccttcccgctct cctcgggaggcccttcctgctctcccctaggctccgcggccgcccagggggtgggag cgggtgaggggagccaggcgcccagcgagagaggccccccgccgcagggcggcccgg gagctcgaggcggtccggcccgcgcgggcagcggcgcggcgctgaggaggggcggcc tggccgggacgcctcggggcgggggccgaggagctctccgggccgccggggaaagct acgggcccggtgcgtccgcggaccagcagcgcgggagagcggactcccctcgccacc gcccgagcccaggttatcctgaatacatgtctaacaattttccttgcaacgttagct gttgtttttcactgtttccaaaggatcaaaattgcttcagaaattggagacatattt gatttaaaaggaaaaacttgaacaaatggacaatatgtctattacgaatacaccaac aagtaatgatgcctgtctgagcattgtgcatagtttgatgtgccatagacaaggtgg agagagtgaaacatttgcaaaaagagcaattgaaagtttggtaaagaagctgaagga gaaaaaagatgaattggattctttaataacagctataactacaaatggagctcatcc tagtaaatgtgttaccatacagagaacattggatgggaggcttcaggtggctggtcg gaaaggatttcctcatgtgatctatgcccgtctctggaggtggcctgatcttcacaa aaatgaactaaaacatgttaaatattgtcagtatgcgtttgacttaaaatgtgatag tgtctgtgtgaatccatatcactacgaacgagttgtatcacctggaattgatctctc aggattaacactgcagagtaatgctccatcaagtatgatggtgaaggatgaatatgt gcatgactttgagggacagccatcgttgtccactgaaggacattcaattcaaaccat ccagcatccaccaagtaatcgtgcatcgacagagacatacagcaccccagctctgtt agccccatctgagtctaatgctaccagcactgccaactttcccaacattcctgtggc ttccacaagtcagcctgccagtatactggggggcagccatagtgaaggactgttgca gatagcatcagggcctcagccaggacagcagcagaatggatttactggtcagccagc tacttaccatcataacagcactaccacctggactggaagtaggactgcaccatacac acctaatttgcctcaccaccaaaacggccatcttcagcaccacccgcctatgccgcc ccatcccggacattactggcctgttcacaatgagcttgcattccagcctcccatttc caatcatcctgctcctgagtattggtgttccattgcttactttgaaatggatgttca ggtaggagagacatttaaggttccttcaagctgccctattgttactgttgatggata cgtggacccttctggaggagatcgcttttgtttgggtcaactctccaatgtccacag gacagaagccattgagagagcaaggttgcacataggcaaaggtgtgcagttggaatg taaaggtgaaggtgatgtttgggtcaggtgccttagtgaccacgcggtctttgtaca gagttactacttagacagagaagctgggcgtgcacctggagatgctgttcataagat ctacccaagtgcatatataaaggtctttgatttgcgtcagtgtcatcgacagatgca gcagcaggcggctactgcacaagctgcagcagctgcccaggcagcagccgtggcagg aaacatccctggcccaggatcagtaggtggaatagctccagctatcagtctgtcagc tgctgctggaattggtgttgatgaccttcgtcgcttatgcatactcaggatgagttt tgtgaaaggctggggaccggattacccaagacagagcatcaaagaaacaccttgctg gattgaaattcacttacaccgggccctccagctcctagacgaagtacttcataccat gccgattgcagacccacaacctttagactgaggtcttttaccgttggggcccttaac cttatcaggatggtggactacaaaatacaatcctgtttataatctgaagatatattt cacttttgttctgctttatcttttcataaagggttgaaaatgtgtttgctgccttgc tcctagcagacagaaactggattaaaacaattttttttttcctcttcagaacttgtc aggcatggctcagagcttgaagattaggagaaacacattcttattaattcttcacct gttatgtatgaaggaatcattccagtgctagaaaatttagccctttaaaacgtctta gagccttttatctgcagaacatcgatatgtatatcattctacagaataatccagtat tgctgattttaaaggcagagaagttctcaaagttaattcacctatgttattttgtgt acaagttgttattgttgaacatacttcaaaaataatgtgccatgtgggtgagttaat tttaccaagagtaactttactctgtgtttaaaaagtaagttaataatgtattgtaat ctttcatccaaaatattttttgcaagttatattagtgaagatggtttcaattcagat tgtcttgcaacttcagttttatttttgccaaggcaaaaaactcttaatctgtgtgta tattgagaatcccttaaaattaccagacaaaaaaatttaaaattacgtttgttattc ctagtggatgactgttgatgaagtatacttttcccctgttaaacagtagttgtattc ttctgtatttctaggcacaaggttggttgctaagaagcctataagaggaatttcttt tccttcattcatagggaaaggttttgtattttttaaaacactaaaagcagcgtcact ctacctaatgtctcactgttctgcaaaggtggcaatgcttaaactaaataatgaata aactgaatattttggaaactgctaaattctatgttaaatactgtgcagaataatgga aacatcacagttcataataggtagtttggatatttttgtacttgatttgatgtgact ttttttggtataatgtttaaatcatgtatgttatgatattgtttaaaattcagtttt tgtatcttggggcaagactgcaaacttttttatatcttttggttattctaagccctt tgccatcaatgatcatatcaattggcagtgactttgtatagagaatttaagtagaaa agttgcagatgtattgactgtaccacagacacaatatgtatgctttttacctagctg gtagcataaataaaactgaatctcaacatacaaagttgaattctaggtttgattttt aagattttttttttcttttgcacttttgagtccaatctcagtgatgaggtaccttct actaaatgacaggcaacagccagttctattgggcagctttgtttttttccctcacac tctaccgggacttccccatggacattgtgtatcatgtgtagagttggtttttttttt ttttaatttttattttactatagcagaaatagacctgattatctacaagatgataaa tagattgtctacaggataaatagtatgaaataaaatcaaggattatctttcagatgt gtttacttttgcctggagaacttttagctatagaaacacttgtgtgatgatagtcct ccttatatcacctggaatgaacacagcttctactgccttgctcagaaggtcttttaa atagaccatcctagaaaccactgagtttgcttatttctgtgatttaaacatagatct tgatccaagctacatgacttttgtctttaaataacttatctaccacctcatttgtac tcttgattacttacaaattctttcagtaaacacctaattttcttctgtaaaagtttg gtgatttaagttttattggcagttttataaaaagacatcttctctagaaattgctaa ctttaggtccattttactgtgaatgaggaataggagtgagttttagaataacagatt tttaaaaatccagatgatttgattaaaaccttaatcatacattgacataattcattg cttcttttttttgagatatggagtcttgctgtgctgcccaggcaggagtgcagtggt atgatctcagctcactgcaacctctgcctcccgggttcaactgattctcctgcctca gcctccctggtagctaggattacaggtgcccgccaccatgcctggctaacttttgta gttttagtagagacggggttttgcctgttggccaggctggtcttgaactcctgacct caagtgatccatccaccttggcctcccaaagtgctgggattacgggcgtgagccact gtccctggcctcattgttcccttttctactttaaggaaagttttcatgtttaatcat ctggggaaagtatgtgaaaaatatttgttaagaagtatctctttggagccaagccac ctgtcttggtttctttctactaagagccataaagtatagaaatacttctagttgtta agtgcttatatttgtacctagatttagtcacacgcttttgagaaaacatctagtatg ttatgatcagctattcctgagagcttggttgttaatctatatttctatttcttagtg gtagtcatctttgatgaataagactaaagattctcacaggtttaaaattttatgtct actttaagggtaaaattatgaggttatggttctgggtgggttttctctagctaattc atatctcaaagagtctcaaaatgttgaatttcagtgcaagctgaatgagagatgagc catgtacacccaccgtaagacctcattccatgtttgtccagtgcctttcagtgcatt atcaaagggaatccttcatggtgttgcctttattttccggggagtagatcgtgggat atagtctatctcatttttaatagtttaccgcccctggtatacaaagataatgacaat aaatcactgccatataaccttgctttttccagaaacatggctgttttgtattgctgt aaccactaaataggttgcctataccattcctcctgtgaacagtgcagatttacaggt tgcatggtctggcttaaggagagccatacttgagacatgtgagtaaactgaactcat attagctgtgctgcatttcagacttaaaatccatttttgtggggcagggtgtggtgt gtaaaggggggtgtttgtaatacaagttgaaggcaaaataaaatgtcctgtctccca gatgatatacatcttattatttttaaagtttattgctaattgtaggaaggtgagttg caggtatctttgactatggtcatctggggaaggaaaattttacattttactattaat gctccttaagtgtctatggaggttaaagaataaaatggtaaatgtttctgtgcctgg tttgatggtaactggttaatagttactcaccattttatgcagagtcacattagttca caccctttctgagagccttttgggagaagcagttttattctctgagtggaacagagt tctttttgttgataatttctagtttgctcccttcgttattgccaactttactggcat tttatttaatgatagcagattgggaaaatggcaaatttaggttacggaggtaaatga gtatatgaaagcaattacctctaaagccagttaacaattattttgtaggtggggtac actcagcttaaagtaatgcatttttttttcccgtaaaggcagaatccatcttgttgc agatagctatctaaataatctcatatcctcttttgcaaagactacagagaataggct atgacaatcttgttcaagcctttccatttttttccctgataactaagtaatttcttt gaacataccaagaagtatgtaaaaagtccatggccttattcatccacaaagtggcat cctaggcccagccttatccctagcagttgtcccagtgctgctaggttgcttatcttg tttatctggaatcactgtggagtgaaattttccacatcatccagaattgccttattt aagaagtaaaacgttttaatttttagcctttttttggtggagttatttaatatgtat atcagaggatatactagatggtaacatttctttctgtgcttggctatctttgtggac ttcaggggcttctaaaacagacaggactgtgttgcctttactaaatggtctgagaca gctatggttttgaatttttagttttttttttttaacccacttcccctcctggtctct tccctctctgataattaccattcatatgtgagtgttagtgtgcctccttttagcatt ttcttcttctctttctgattcttcatttctgactgcctaggcaaggaaaccagataa ccaaacttactagaacgttctttaaaacacaagtacaaactctgggacaggacccaa gacactttcctgtgaagtgctgaaaaagacctcattgtattggcatttgatatcagt ttgatgtagcttagagtgcttcctgattcttgctgagtttcaggtagttgagataga gagaagtgagtcatattcatattttcccccttagaataatattttgaaaggtttcat tgcttccacttgaatgctgctcttacaaaaactggggttacaagggttactaaatta gcatcagtagccagaggcaataccgttgtctggaggacaccagcaaacaacacacaa caaagcaaaacaaaccttgggaaactaaggccatttgttttgttttggtgtcccctt tgaagccctgccttctggccttactcctgtacagatatttttgacctataggtgcct ttatgagaattgagggtctgacatcctgccccaaggagtagctaaagtaattgctag tgttttcagggattttaacatcagactggaatgaatgaatgaaactttttgtccttt ttttttctgtttttttttttctaatgtagtaaggactaaggaaaacctttggtgaag acaatcatttctctctgttgatgtggatacttttcacaccgtttatttaaatgcttt ctcaataggtccagagccagtgttcttgttcaacctgaaagtaatggctctgggttg ggccagacagttgeactetctagtttgccctctgccacaaatttgatgtgtgacctt tgggcaagtcatttatcttctctgggccttagttgcctcatctgtaaaatgagggag ttggagtagattaattattccagctctgaaattctaagtgaccttggctaccttgca gcagttttggatttcttccttatctttgttctgctgtttgagggggctttttactta tttccatgttattcaaaggagactaggcttgatattttattactgttcttttatgga caaaaggttacatagtatgcccttaagacttaattttaaccaaaggcctagcaccac cttaggggctgcaataaacacttaacgcgcgtgcgcacgcgcgcgcgcacacacaca cacacacacacacacacacacaggtcagagtttaaggctttcgagtcatgacattct agcttttgaattgcgtgcacacacacacgcacgcacacactctggtcagagtttatt aaggctttcgagtcatgacattatagcttttgagttggtgtgtgtgacaccaccctc ctaagtggtgtgtgcttgtaattttttttttcagtgaaaatggattgaaaacctgtt gttaatgcttagtgatattatgctcaaaacaaggaaattcccttgaaccgtgtcaat taaactggttcatatgactcaagaaaacaataccagtagatgattattaactttatt cttggctctttttaggtccattttgattaagtgacttttggctggatcattcagagc tctcttctagcctacccttggatgagtacaattaatgaaattcatattttcaaggac ctgggagccttccttggggctgggttgagggtggggggttggggagtcctggtagag gccagctttgtggtagctggagaggaagggatgaaaccagctgctgttgcaaaggct gcttgtcattgatagaaggactcacgggcttggattgattaagactaaacatggagt tggcaaactttcttcaagtattgagttctgttcaatgcattggacatgtgatttaag ggaaaagtgtgaatgcttatagatgatgaaaacctggtgggctgcagagcccagttt agaagaagtgagttgggggttggggacagatttggtggtggtatttcccaactgttt cctcccctaaattcagaggaatgcagctatgccagaagccagagaagagccactcgt agcttctgctttggggacaactggtcagttgaaagtcccaggagttcctttgtggct ttctgtatacttttgcctggttaaagtctgtggctaaaaaatagtcgaacctttctt gagaactctgtaacaaagtatgtttttgattaaaagagaaagccaactaaaaaaaaa aaaaaaaaaaa 21 Mouse SMAD4- ccgctgcggtaacggagcggctcgggcggcggagcccgtgttcgcgtccgtccgccc NM_008540.2 gcccgcccgccgtcctccggaggcccttcccgcgccgcgctccgctccgcggccgtc cccggggcgggagcgcgtgaccggagccggcgcccgcgagcgaggccccccgcagcg gggcggctccggagctccagcggcccggccggccggcgcggtccgcggcgcggcggg gagagggggccgcctgggccggacgccgcgggcggggcccgggaagcgacagcgagg cgaggcgcggtgcggcgcggagcccaggtcatcctgctcaccagatgtcttgacagt ttttcttgcaacattggccattggttttcactgccttcaaaagatcaaaattactcc agaaatcggagagttggatttaaaagaaaaaacttgaacaaatggacaatatgtcta taacaaatacaccaacaagtaacgatgcctgtctgagcattgtacatagtttgatgt gccatagacaaggtggggaaagtgaaacctttgcaaaaagagcaattgagagtttgg taaagaagctgaaagagaaaaaagatgaattggattctttaataacagctataacta caaatggagctcatcctagcaagtgtgtcaccatacagagaacattggatggacgac ttcaggtggctggtcggaaaggatttcctcatgtgatctatgcccgtctgtggaggt ggcctgatctacacaagaatgaactaaagcatgttaaatattgtcagtatgcgtttg acttaaaatgtgacagtgtctgtgtgaatccatatcactatgagcgggttgtctcac ctggaattgatctctcaggattaacactgcagagtaatgctccaagtatgttagtga aggatgagtacgttcacgactttgaaggacagccgtccttacccactgaaggacatt cgattcaaaccatccaacacccgccaagtaatcgcgcatcaacggagacgtacagcg ccccggctctgttagccccggcagagtctaacgccaccagcaccaccaacttcccca acattcctgtggcttccacaagtcagccggccagtattctggcgggcagccatagtg aaggactgttgcagatagcttcagggcctcagccaggacagcagcagaatggattta ctgctcagccagctacttaccatcataacagcactaccacctggactggaagtagga ctgcaccatacacacctaatttgcctcaccaccaaaacggccatcttcagcaccacc cgcctatgccgccccatcctggacattactggccagttcacaatgagcttgcattcc agcctcccatttccaatcatcctgctcctgagtactggtgctccattgcttactttg aaatggacgttcaggtaggagagacgtttaaggtcccttcaagctgccctgttgcga ctgtggatggctatgtggatccttcgggaggagatcgcttttgcttgggtcaactct ccaatgtccacaggacagaagcgattgagagagcgaggttgcacataggcaaaggag tgcagttggaatgtaaaggtgaaggtgacgtttgggtcaggtgccttagtgaccacg cggtctttgtacagagttactacctggacagagaagctggccgagcacctggcgacg ctgttcataagatctacccaagcgcgtatataaaggtctttgatctgcggcagtgtc accggcagatgcagcaacaggcggccactgcgcaagctgcagctgctgctcaggcgg cggccgtggcagggaacatccctggccctgggtccgtgggtggaatagctccagcca tcagtctgtctgctgctgctggcatcggtgtggatgacctccggcgattgtgcattc tcaggatgagctttgtgaagggctggggcccagactaccccaggcagagcatcaagg aacccccgtgctggattgagattcaccttcaccgagctctgcagctcttggatgaag tcctgcacaccatgcccattgcggacccacagcctttagactgagatctcacaccac ggacgccctaaccatttccaggatggtggactatgaaatatactcgtgtttataatc tgaagatctattgcattttgttctgctctgtcttttcctaaagggttgagagatgtg tttgctgccttgctcttagcagacagaaactgaattaaaacttcttttctattttag aactttcaggtgtggctcagtgcttgaagatcagaaagatgcagttcttgctgagtc ttccctgctggttctgtatggaggagtcggccagtgctgggcgctcagccctttagt gtgtgcgagcgccttgcatgccgaggagagtcagagctgctgattgtaaggctgaga agttctcacagttaagccacctgccccttagtgggcgagttattaaacgcactgtgc tcacgtggcgctgggccagccagctctaccaagagcaactttactctcctttaaaaa ccttttagcaacctttgattcacaatggtttttgcaagttaaacagtgaaggtgaat taaattcatactgtcttgcagacttcagggtttcttccccaagacaaaacactaatc tgtgtgcatattgacaattccttacaattatcagtcaaagaaatgccatttaaaatt acaatttttttaatccctaatggatgaccactatcaagatgtatactttgccctgtt aaacagtaaatgaattcttctatatttctaggcacaaggttagttatttaaaaaaaa aaaaaaaagcctaggggagggatttttcccttaattcctagggagaaggttttgtat aaaacactaaaagcagcgtcactctgcctgctgcttcactgttctgcaaggtggcag tacttcaactgaaataatgaatattttggaaactgctaaattctatgttaaatactg tgcagaataatggaaacagtgcagttggtaacaggtggtttggatatttttgtactt gatttgatgtgtgacttcttttcatatactgttaaaatcatgtatgttttgacattg tttaaaattcagtttttgtatcttagggcaagactgcagacttttttataccttttg gttataagccctgtgtttgccatccttgatcacttggcggtgactttgtagagattg aagtggaggagttaagacacattgactgtaccacagacacacatgcatactttctac ctagttactagcgtaaataaaactgagtcactataaaaaaaaaaaaaaaaaaaaa 22 Mouse IL6R- gcagtgcgagctgagtgtggagcccgaggccgagggcgactgctctcgctgccccag NM_010559.2 tctgccggccgcccggccccggctgcggagccgctctgccgcccgccgtcccgcgta gaaggaagcatgctgaccgtcggctgcacgctgttggtcgccctgctggccgcgccc gcggtcgcgctggtcctcgggagctaccgcgcgctggaggtggcaaatggcacagtg acaagcctgccaggggccaccgttaccctgatttgccccgggaaggaagcagcaggc aatgttaccattcactgggtgtactctggctcacaaaacagagaatggactaccaca ggaaacacactggttctgagggacgtgcagctcagcgacactggggactatttatgc tccctgaatgatcacctggtggggactgtgcccttgctggtggatgttcccccagag gagcccaagctctcctgcttccggaagaacccccttgtcaacgccatctgtgagtgg cgtccgagcagcaccccctctccaaccacgaaggctgtgctgcttgcaaagaaaatc aacaccaccaacgggaagagtgacttccaggtgccctgtcagtattctcagcagctg aaaagcttctcctgccaggtggagatcctggagggtgacaaagtataccacatagtg tcactgtgcgttgcaaacagtgtgggaagcaagtccagccacaacgaagcgtttcac agcttaaaaatggtgcagccggatccacctgccaaccttgtggtatcagccatacct ggaaggccgcgctggctcaaagtcagctggcagcaccctgagacctgggacccgagt tactacttgctgcagttccagcttcgataccgacctgtatgttcaaaggagttcacg gtgttgctgctcccggtggcccagtaccaatgcgtcatccatgatgccttgcgagga gtgaagcacgtggtccaggtccgtgggaaggaggagcttgaccttggccagtggagc gaatggtccccagaggtcacgggcactccttggatagcagagcccaggaccaccccg gcaggaatcctctggaaccccacacaggtctctgttgaagactctgccaaccacgag gatcagtacgaaagttctacagaagcaacgagtgtcctcgccccagtgcaagaatcc tcgtccatgtccctgcccacattcctggtagctggaggaagcttggcgtttgggttg cttctctgtgtcttcatcatcctgagactcaagcagaaatggaagtcagaggctgag aaggaaagcaagacgacctctcctccacccccaccgtattccttgggcccactgaag ccgaccttccttctggttcctctcctcaccccacacagctctgggtctgacaatacc gtaaaccacagctgcctgggtgtcagggacgcacagagcccttatgacaacagcaac agagactacttattccccagacaatcatctggatggtacctggcagctggcagggca ccacgagatcagcacacaagtttctcatgcgggtcccatccacctggggtggggtgg ggcgggcggggctgcagcttcactaacccacaagagctctgcacaggttctgagtag gtgcagctggtgctgcataggctctgaaggaaggaaggggctgtgaggaacacaggc cattgcgaagacagcttgtgatgactgaatagagatgcccgtcagctccacatctga tagtggctcacaagctgcaccctcaggaggcctcagaaaggggctccaaaggctgcc ccagctgcctcgctctgcctcactgccccaagccaccttttagctctcgaactccta aagtccaagcactttgccattctctttccgaggccactgaggccgggtggaagcttg gttccgatttccttctcaacatctggaaagcagctgggcccggtggtggtgactaat atctcagggcctgatggtttacgcgagtgacaatttcccacaagcagtttttaaatg tgaatgatgaccccaggcactgctggctgcggaggcttcattttcctcttcgatctc aggacttcaggcgaaaagcggagtggaagtagagagcggatgggtgtccaccgtcct catggtacttgcgggaggtacagcctggaaaacacgtttcctgtccccctactctcc caggagagggatgatggtagggggtgcctcttccagggcggagagaactactttacc ccagccttgcccattctgatttcaactggactggagctactaggaaagtcgacattc atgcaaaaagaaaaaacgttaactagcaagaatgcactttcattttggtttttagag aactgttgcctgtttctctcaagagtctggaagaggccgctcactgcacactactgt atgaaccctcactgcccaccctggaggaccaagtgcagtaacggtagcccaaacacc aagtcaagtgaaaatcgagggaaaaaaaaaacaaacaagcaacaaaaaaaaaaaacc aaaactaaactaaaaaacaaatcacccccccaaaaaaaaacaaaaccaaaaaccaaa aaaaacaaaaaaacaaaacaacaacaacaaaaaaaacccaaaccaacccgctgtttc ctataacagaaaagcctttggtttcattttttattttgatttttttgtcttaaaaag tataaaaatagcctgtccatgctctgcttcagggaatgagcctgtgaacactcccag gcgcaggcaggaagggtgtctgcttcctgctacacctcactgccaccttggccttcc ttgctttacgtttgactgagtggcctcagacgctttcccctggggccttgaggaatc cagtgatgttagtggtcaccgaggagaccacagagccacagtgtggtgcttagatta aagtgacttctgcaaccacagcaccccacacctgccgtcttactgaactacgccagt aacttgccttttctgccaccaccacgagacgagacgggcagagctcggaagctgtca ccccatgccctctgcttgtccgctctaggggccactgacctaagcattagttatttt attttattttatttttttgtgggttttgtacattttaggtcctgttgctgtcttaga aaaggctctgtaggttgacagaaaatcaggccaagtattcatgttttgttttttttt tttttccttctttcctcctttgctaagtttttgggactcaagggtagcaaaactgct gtgaaagggaaatttattaaaaatgttacagatcgtg 23 Rat IL6R- gccccacgtagaaggaaccatgctggccgtcggctgcaccctgctggtcgccctgct NM_017020.3 ggccgcgcccgcagtcgcgctggtccttgggagctgccgcgcgctggaggcggcaaa tggtacggtgacgagcctgccaggggccactgttaccctgatctgccctgggaagga agcagcaggcaatgctaccattcactgggtgtactcaggctcacagagcagagaatg gactaccacgggaaacacactggttctgagggccgtgcaggtcaacgacactgggca ctatttgtgcttcctggatgatcatctggttgggactgtgcccttgctggtggatgt tcccccagaggagcccaagctctcctgcttccggaagaacccccttgtaaatgcctt ttgtgagtggcatccaagcagcactccctctccaaccacgaaggctgtgatgtttgc aaagaaaatcaacaccaccaatgggaagagtgacttccaggtgccttgccagtattc tcagcagctgaaaagcttctcctgcgaggtggagatcctggagggtgacaaagtgta ccacatagtgtcactgtgcgttgcaaacagtgtcggaagcaggtccagccacaatgt agtatttcagagtttaaaaatggtgcagccggatccacctgccaaccttgtggtatc agccatacctggaaggcctcgttggctcaaagtcagttggcaagaccctgagtcctg ggacccaagttactacttgttgcaattcgagcttcgataccgacctgtatggtcaaa gacgttcacggtgtggccgctccaggtggcccagcatcaatgtgtcatccatgatgc cttgcgaggagtaaagcatgtggtgcaggtccgagggaaggaggagtttgacattgg ccagtggagcaaatggcccccggaggtcacaggcactccttggctagcagagcccag gaccactccggcagggatcccggggaaccccacacaggtctctgttgaagactatga caaccacgaggatcagtacggaagttctacagaagcaacgagtgtcctcgccccagt gcaaggatcctcgcctatacccctgcccacattcctggtagctggaggaagcctggc gtttggattgcttctctgtgtcttcatcatcttgagactcaagaagaaatggaagtc acaggctgagaaggaaagcaagacgacttctcccccaccgtatcccttgggaccgct gaagccgaccttcctcctggttcctctccctaccccatcagggtcccataacagctc tgggactgacaacaccggaagccacagctgcctgggtgtcagggacccacagtgccc taatgacaacagcaacagagactacttattccccagataattgtctggagggtacct ggcagctggcacgcaagtttctcactgccggccccgtccaccagggctgggggcggg gtgggcggggctgcagcttcacgatcccacaggagccttgcaaaggttctgagtggg agaagactggtgtgctgcacgggcttcgaaagaaggggctgtgaggagcacgagcca tcatgaagagagcccgtgatgactctgaatagagacgcccgcccatcagctacacac ctgatggtggctctcaagctatcctctcaggaagcctctgggaggggcgacaaaggc tgccccagttgcctagctctggctcactggcccaagctgccttttagcttgaactcc taaaatccaagcaccttggccattctcttcctaggccaccgaggccgcggggaagct tggttctactttccttctcaacacctggagaagcagctgcccggtggtggtgactaa cgtatcagggcctgatggcttatgaggaatgacaattaattcctcataagcagtttt taaatgtgaatagtaatcctaggcactgctgacttgaggttttattttcttcaatct caggacttcaggagagaagcagagcagaagtagagagaggatgggtgtccattgtcc gtgtggtacttgagggggatacagcctggaaaacacgtttcctgcccccctactctc ccagaagaggtagggggtggcgcctcttccagggcagagagtataactactttacct ggccttgcccatactggtttcaactggacttgagctactaggaaaaatgacattcat gcaaaaagaaaactttaactagcaagaatgcacttccactttggtttctagaggact gttgctcctcttgagacgctggaagaggccgctcactgtaccctggtgtatgagccc tcaccccccaccccagggtaagtgcagtaactttagtctaaacaccgagtcaggtaa aaatcgaggaaaaaacaaccccgtttcctgtaacagaaaagcctttggtttcgtttt gtattttgattttttttttgtcttaaaaagtgtaaaaatagtctgtccatactctgc ttcagggaatgacctgtgaatactccccaggcgtgggcaggaagggtgtctgcttcc tgctacacctcactgccacctcggccttccttgctttacattcaactgagttgcctc agctgctttcccctggggcgctgaaaaagccagtgatgttggtggtcaccgagaaga ccacagagccacagagtaatgctgtgattgaagcgagctacgcaaccacagcacccc acatttgctgtattatagaactatgctaggagcttgccctttcacaaaataccacca ccacgagacgtggcagagctcggaagctgtcaccttgtgccatctgcttgccagctc caaggggccactgacttaagcagttattttctttgtgggctttgttcatttcagggc ctgttgctgtcttagaaaaagctctgtcggttgacaaaaacatcagacaggtagtca tgtttatttattttttttcctcttttgctaagtctttgggactcaagggtagtaaaa aatgctgtgaaaagggaaacattagaaacagcgatcttcggggaataggtgactgtg cccacgcactgttcttcagtccctcacgtggctctgcccgagcgctgttccaagcca ggcagagcaggctggcggaagattgaaatccagatagttcgttatctctgagagcta aatagctttgatctccaagctgttattgctttcactattgtaacaggatagcctccc ccccccatgtcaaaaggatgcttttcccttttgactttttataagctaagtcagtga agtctgtttcatctgagctccagcttcgttcagttcgcacaggtgtatgccctcagc tgcttcgggcctcagatctgtgctagttgaatggttgtcccatccttgggtcatcct taccagagtttctgcagcccacaggtctgccttgtcaacagtaccacttaacaccag cattcagtgcccaggcagccagatgtggagggtttacccagagatgatttaaacatg accttaaacgtgtatggtagaacgaggggaacccataccagctcaggttctaaagag atctttgattcttctggcattagtgaaatagctttaaactatttcaaggaagaagcc ttggccacacccacgacatttggtgacaatcctttctctccatgagccttgtcttta caccttctcacctggctgaaagctcacactgaatctttcctatgcccctggtgtctt gggagaaaggaaactggtatgggcttcactgctggaattggcttggagccagcgtgt ggcgcagcgcctggcagggtgggccaggcttagttatggtgtgctggtttaaggaat gcctggcttgcctggttgcttgggttctgagctgcagagtttcctagcagttcttta tggctgacctagttggggaagattcccacactcaactgcaggtggaggtggtgagaa agctgttttcattcggagaggcaggatcagcccaagaagctttcagtgggagagcct acagtgaggctgtacctcactgtgggaggaggcaggccagctggctcaggtcctggg actggcactggggagggtctgccaaaggtccctccagcctgtagtcctagcatagtc gggtgccagttccaggaagtttctacggcaaccttagtgctcattaaggaacattgt cagttttgtgaacatatgctcagatggagatcttgctttcagagaaaggactggtac agtgtgtaacaagctggagcagacagagagactttttggcaagagatcacatccgtt aagcagaatacctcagtgctacatgtttttgtctttgagacaatgtttttaaggttt ttatgctctgttacctgtaagctgatacctaaaactttctgcaaagtcagggttttt caatgccttttttttttttttgccattgtttgctttaaagtgaagattgtaactgtt tgaaataaataatttctaaaactgca 24 Human BMP6- caactgggggcgccccggacgaccatgagagataaggactgagggccaggaagggga NM_001718.4 agcgagcccgccgagaggtggcggggactgctcacgccaagggccacagcggccgcg ctccggcctcgctccgccgctccacgcctcgcgggatccgcgggggcagcccggccg ggcggggatgccggggctggggcggagggcgcagtggctgtgctggtggcgggggct gctgtgcagccgctgcgggcccccgccgctgcggccgcccttgcccgctgccgcggc cgccgccgccggggggcagctgctgggggacggcgggagccccggccgcacggagca gccgccgccgtcgccgcagtcctcctcgggcttcctgtaccggcggctcaagacgca ggagaagcgggagatgcagaaggagatcttgtcggtgctggggctcccgcaccggcc ccggcccctgcacggcctccaacagccgcagcccccggcgctccggcagcaggagga gcagcagcagcagcagcagctgcctcgcggagagccccctcccgggcgactgaagtc cgcgcccctcttcatgctggatctgtacaacgccctgtccgccgacaacgacgagga cggggcgtcggagggggagaggcagcagtcctggccccacgaagcagccagctcgtc ccagcgtcggcagccgcccccgggcgccgcgcacccgctcaaccgcaagagccttct ggcccccggatctggcagcggcggcgcgtccccactgaccagcgcgcaggacagcgc cttcctcaacgacgcggacatggtcatgagctttgtgaacctggtggagtacgacaa ggagttctcccctcgtcagcgacaccacaaagagttcaagttcaacttatcccagat tcctgagggtgaggtggtgacggctgcagaattccgcatctacaaggactgtgttat ggggagttttaaaaaccaaacttttcttatcagcatttatcaagtcttacaggagca tcagcacagagactctgacctgtttttgttggacacccgtgtagtatgggcctcaga agaaggctggctggaatttgacatcacggccactagcaatctgtgggttgtgactcc acagcataacatggggcttcagctgagcgtggtgacaagggatggagtccacgtcca cccccgagccgcaggcctggtgggcagagacggcccttacgacaagcagcccttcat ggtggctttcttcaaagtgagtgaggtgcacgtgcgcaccaccaggtcagcctccag ccggcgccgacaacagagtcgtaatcgctctacccagtcccaggacgtggcgcgggt ctccagtgcttcagattacaacagcagtgaattgaaaacagcctgcaggaagcatga gctgtatgtgagtttccaagacctgggatggcaggactggatcattgcacccaaggg ctatgctgccaattactgtgatggagaatgctccttcccactcaacgcacacatgaa tgcaaccaaccacgcgattgtgcagaccttggttcaccttatgaaccccgagtatgt ccccaaaccgtgctgtgcgccaactaagctaaatgccatctcggttctttactttga tgacaactccaatgtcattctgaaaaaatacaggaatatggttgtaagagcttgtgg atgccactaactcgaaaccagatgctggggacacacattctgccttggattcctaga ttacatctgccttaaaaaaacacggaagcacagttggaggtgggacgatgagacttt gaaactatctcatgccagtgccttattacccaggaagattttaaaggacctcattaa taatttgctcacttggtaaatgacgtgagtagttgttggtctgtagcaagctgagtt tggatgtctgtagcataaggtctggtaactgcagaaacataaccgtgaagctcttcc taccctcctcccccaaaaacccaccaaaattagttttagctgtagatcaagctattt ggggtgtttgttagtaaatagggaaaataatctcaaaggagttaaatgtattcttgg ctaaaggatcagctggttcagtactgtctatcaaaggtagattttacagagaacaga aatcggggaagtggggggaacgcctctgttcagttcattcccagaagtccacaggac gcacagcccaggccacagccagggctccacggggcgcccttgtctcagtcattgctg ttgtatgttcgtgctggagttttgttggtgtgaaaatacacttatttcagccaaaac ataccatttctacacctcaatcctccatttgctgtactctttgctagtaccaaaagt agactgattacactgaggtgaggctacaaggggtgtgtaaccgtgtaacacgtgaag gcaatgctcacctcttctttaccagaacggttctttgaccagcacattaacttctgg actgccggctctagtaccttttcagtaaagtggttctctgcctttttactatacagc ataccacgccacagggttagaaccaacgaagaaaataaaatgagggtgcccagctta taagaatggtgttagggggatgagcatgctgtttatgaacggaaatcatgatttccc ttgtagaaagtgaggctcagattaaattttagaatattttctaaatgtctttttcac aatcatgtactgggaaggcaatttcatactaaactgattaaataatacatttataat ctacaactgtttgcacttacagctttttttgtaaatataaactataatttattgtct attttatatctgttttgctgtaacattgaaggaaagaccagacttttaaaaaaaaag agtttatttagaaagtatcatagtgtaaacaaacaaattgtaccactttgattttct tggaatacaagactcgtgatgcaaagctgaagttgtgtgtacaagactcttgacagt tgcgcttctctaggaggttgggtttttttaaaaaaagaattatctgtgaaccatacg tgattaataaagatttcctttaaggca 25 Rhesus BMP6- agcgcagcccggactcggacgcacctggcctgtaccgcgcgcctctagagacctgcg XM_001085364 cggggctgtggggctccccttcctcccctccaagcggttctcccggtgatcgcccct tcgccacccctctatcctgggcaactgggggcgccccggacgaccatgagagataag gactgagggccaggaaggggaagcgagcccgccgagaggtggcgggggctgctcacg ccaagggccacagcggccgtgctccagcctcgctccgccgctccacgcctcgcggga tccgcgggggcagcccggccgggcggggatgccggggctggggcggagggcgcagtg gctgtgctggtggtgggggctgttgtgcagctgctgcgggcccccgccgctgcggcc gcccctgcccgctgccgcggccgccgccgccgggggccagctgctgggggacggcgg gagccccggccgcacggagcagccgccgccgtcgccgcaatcctccccgggcttcct ctaccggcggctcaagacgcacgagaagcgggagatgcagaaggagatcttgtcggt gctggggctcccacaccggccccggcccctgcacggcctccaacagccgcagccccc ggcgctcccgcagcagcagcagcagcagcagcagccgcctcgcggagagccccctcc cgggcagctgaagcccgcgcccctcttcatgctggatctgtacaacgccctgtccgc cgacgacgaggaggacggggcgtcggagggggagaggcagcagccctggccccacga aggagccagctcgtcccagcctcggcagccggccccgggcgccgcgcacccgctcaa ccgcaagagcctcctggcccccggacctggcagcggcggcgcgtccccactgaccag cgcgcaggacagcgccttcctcaacgacgcagacatggtcatgagctttgtgaacct ggtggagtacgacaaggagttctcccctcgtcagcgacaccacaaagagttcaagtt caacttatcccagattcctgagggtgaggcggtgacggctgcagaattccgcatcta caaggactgtgttgtggggagttttaaaaaccaaacttttcttatcagcatttatca agtcttacaggagcatcagcacagagactctgacctttttttgctggacacccgcgt agtgtgggcctcagaagaaggctggctggaatttgacatcacggccactagcaatct gtgggccgtgaccccgcagcataacatggggcttcagctgagtgtggtgacgcggga tggagtccacatccatccccgagccgcgggcctggtgggcagagacggcccttacga caagcagcccttcatggtggctttcttcaaagtgagtgaggtccacgtgcgcaccac caggtcagcctctggctggcgccgacaacagagtcgtaatcgctctacccagtccca ggacgtggcgcgggtctccagtgcttcagattacaacagcagtgaattaaaaacagc ttgcaggaagcatgagctgtatgtgagtttccaagacctgggatggcaggactggat cattgcacccaagggctacgctgccaattactgtgatggggaatgctccttcccact caacgcacacatgaatgcaaccaaccacgcgatcgtgcagaccttggttcaccttat gaaccctgagtatgtccccaaaccgtgctgtgcgccaactaaactaaatgccatctc agttctttactttgatgacaattccaatgtcattctgaaaaaatacaggaatatggt tgtaagagcttgtggatgccactaactcgaaaccagatgctggggacacacattctg ccttggattcctagattacatctgccttaaaaaacacagaagcacagttggaggtgg gacgatgagacttggaaactatctcatgccagtgccttattacccaagaagatttta aaggacctcattaataatttgctcacttggtaaatgacgtgagtagttgttggtctg tagcaagctgagtttggatgtctgtagtgcaaggtccggtaactgcagaaagcaccg tgaagctcttcctcccctcctcccccaaaaacccaccaaaattagttttagctgtag atcaagctatttggggtgttagtaagtagggaaaataatctcaaaggagttaaatgt attcttggttaaagtatcagcctgttcagtactgtctatcaaaggtagattttacag agaacagaaattggggaagttgggggaacgcctctgttcagatttcattcccaggaa gttcaacttcatacatgacccacagcccaggccacagccagggctccatggggcgcc tttgtctcagtcattgctgttatgtgttcatgctggagttttgttg 26 Mouse BMP6- gatcctggccgtcgccccgtcgtctcttctccacccgggcttctgggggcgccgcgg NM_007556.2 atgaccatgagagataaggactgagtgccaggaccgggaagagagcccgccgagagg tggcgggggctgcccactccgagggccacagcctccgcgctccggcctcgctccgcc gctcgacgcctcgcgggccccgcgggggcagccgggctgggcggcgatgcccgggct ggggcggagggcgcagtggctgtgctggtggtgggggttgctgtgcagctgcggccc cccgccactgcggccccctctgccggtagccgcggccgccgccggggggcagctgct gggagccggcgggagcccggtgcgcgctgagcagccaccgccacagtcctcttcttc gggcttcctctatcggcggctcaagacccacgagaagcgggagatgcaaaaggagat cctgtcggtgctggggctcccgcacaggccgcggcccctgcacggtctccagcagcc tcagcccccggtactcccgccacagcagcagcagcagcagcagcagcagcagacggc ccgcgaggagccccctccagggcggctgaagtccgctccactcttcatgctggatct ctacaacgccctgtccaatgacgacgaagaggatggggcatcggagggtgtggggca agagcctgggtcccacggaggggccagctcgtcccagctcaggcagccgtctcccgg cgctgcacactccttgaaccgcaagagtctcctggccccgggacccggtggcggtgc gtccccactgactagcgcgcaggacagcgctttcctcaacgacgcggacatggtcat gagctttgtgaacctggtggagtacgacaaggagttctccccacatcaacgacacca caaagagttcaagttcaacctatcccagattcctgagggtgaggcggtgacggctgc tgagttccgcgtctacaaggactgtgtggtggggagttttaaaaaccaaacctttct tatcagcatttaccaagtcttgcaggagcatcagcacagagactctgacctattttt gttggacacccgggtggtgtgggcctcagaagaaggttggctggaatttgacatcac agcaactagcaatctgtgggtggtgacaccgcagcacaacatggggctccagctgag tgtggtgactcgggatggactccacgtcaacccccgtgcggcgggcctggtgggcag agacggcccttacgacaagcagcccttcatggtggccttcttcaaggtgagcgaggt ccacgtgcgcaccaccaggtcagcctccagtcggcggcggcagcagagtcgcaaccg gtccacccagtcgcaggacgtgtcccggggctccggttcttcagactacaacggcag tgagttaaaaacagcttgcaagaagcatgagctctatgtgagcttccaggacctggg atggcaggactggatcattgcacccaaaggctacgctgccaactactgtgatggaga gtgttccttcccactcaacgcacacatgaatgccaccaaccacgccattgtacagac cttggtccaccttatgaatcccgagtacgtccccaaaccatgctgcgcaccaaccaa actgaatgccatctcggttctttacttcgatgataactccaatgtcatcttgaaaaa gtacaggaatatggtcgtgagagcttgtggttgccattaagttgaagctggtgtgtg tgtgtgggtgggggcatggttctgccttggattcctaacaacaacatctgccttaaa ccacgaacaacagcacagcgaagcgggatggtgacacacagagggatcgtgacacgc agacacatctcccgctggtgccttacccacggaggcttttatgaggaccttgtcaag ggctttcccagttcctaactgagcagttgctggtctgcaggaagctggaaggcttgt agtacaggcctggaaactgcagttacctaatgttcgcctcccccaaccccgcccgga gtagttttagcttttagatctagctgcttgtggtgtaagtagagagtaaacttgaag gaatattaaatatccctgggttgaaagacccggtggtggctctacagcaeccatccc agggagatttttgcagacatccgaatggaggggagaagggcactctttcaggttcca ttcccagcaagggcagctcacacaggacctgcagcctggccatcagcaggctctgtg gaggtgccttctgtctactgttgtagttacgtgttttgtgttgactctcggtggtgt gagaatgtactaatctctgtcaagacaaactgtagcatttccaccccatcctcctcc ctccctcacagaattc 27 Rat BMP6- atgcccgggctggggcggagggcgcagtggctgtgctggtggtgggggtcactgtgc NM_013107.1 agctgcggccccccgccactgcggccccctctgccggtagccgcggccgccgccggg gggcagctgctgggagccggcgggagccctgtgcgcgccgagcagccaccgccgcaa tcctcctcttcgggcttcctctatcggcggctcaagacccacgagaagcgggagatg caaaaggagatcctgtcggtgttggggctgcctcacaggccgcggcccctgcacggt ctccagcagcctcaatcccccgtgctcccgcagcagcaacaatcgcaacagacggcc cgcgaggagccccctccagggcggctgaagtccgctccgctcttcatgctggatctc tacaactccctgtccaaggacgacgaagaggatggggtgtcagagggagagggactg gagcccgagtcccacggaagggccagctcgtcccagctcaaacagccatctcccggg gctgcacactccctgaaccgcaagagtctcctggccccgggacccggcggcagtgcg tccccactgaccagcgcgcaggacagcgctttcctcaacgacgcggacatggtcatg agctttgtgaacctggtggagtacgacaaggagttctccccacgccagcgacaccac aaggagttcaagttcaacttatcccagattcccgagggtgaggcagtgacggctgca gagttccgcgtctacaaggactgtgtggtggggagttttaaaaaccaaacttttctt atcagcatttaccaagtcttacaggagcatcagcacagagactctgacctatttttg ttggacacccgggtggtgtgggcctccgaagaaggctggctggaattcgacatcaca gcaactagcaatctgtgggtggtgacaccgcagcacaacatgggactccagctgagt gtggtgactcgggacggactccacatcaacccccgtgcggcgggcctggtgggcaga gacggcccttacgacaagcagcccttcatggtggccttcttcaaggcgagcgaggtc cacgtgcgcaccaccaggtcagcctccagtcggcgtcgacagcagagtcgcaatcgg tccacccagtcgcaggacgtgtcccggggctccagtgcttcagactacaacagcagt gagttaaaaacagcttgcaagaagcatgagctttacgtgagcttccaggacctggga tggcaggactggatcatcgcacccaaaggctacgctgccaactattgtgacggagag tgttccttccctctcaatgcacacatgaatgccaccaaccacgccattgtacagacc ttggtccaccttatgaatcccgagtacgtccccaaaccatgctgcgcaccaaccaaa ctgaatgccatctcggttctttacttcgacgacaactccaatgtcatcttgaaaaaa tacaggaacatggttgtgagagcttgtggatgtcattga 28 Human NEO1- gggccgggccgggctgggctggagcagcggcggccgcgggagccgagcttgcagcga NM_002499.2 gggaccggctgaggcgcgcgggagggaaggaggcaagggctccgcggcgctgtcgcc gccgctgccgctcactctcggggaagagatggcggcggagcggggagcccggcgact cctcagcaccccctccttctggctctactgcctgctgctgctcgggcgccgggcgcc gggcgccgcggccgccaggagcggctccgcgccgcagtccccaggagccagcattcg aacgttcactccattttattttctggtggagccggtggatacactctcagttagagg ctcttctgttatattaaactgttcagcatattctgagccttctccaaaaattgaatg gaaaaaagatggaacttttttaaacttagtatcagatgatcgacgccagcttctccc ggatggatctttatttatcagcaatgtggtgcattccaaacacaataaacctgatga aggttattatcagtgtgtggccactgttgagagtcttggaactattatcagtagaac agcgaagctcatagtagcaggtcttccaagatttaccagccaaccataaccttcctc agtttatgctgggaacaatgcaattctgaattgcgaagttaatgcagatttggtccc atttgtgaggtgggaacagaacagacaaccccttcttctggatgatagagttatcaa acttccaagtggaatgctggttatcagcaatgcaactgaaggagatggcgggcttta tcgctgcgtagtggaaagtggtgggccaccaaagtatagtgatgaagttgaattgaa ggttcttccagatcctgaggtgatatcagacttggtatttttgaaacagccttctcc cttagtcagagtcattggtcaggatgtagtgttgccatgtgttgcttcaggacttcc tactccaaccattaaatggatgaaaaatgaggaggcacttgacacagaaagctctga aagattggtattgctggcaggcggtagcctggagatcagtgatgttactgaggatga tgctgggacttatttttgtatagctgataatggaaatgagacaattgaagctcaagc agagcttacagtgcaagctcaacctgaattcctgaagcagcctactaatatatatgc tcacgaatctatggatattgtatttgaatgtgaagtgactggaaaaccaactccaac tgtgaagtgggtcaaaaatggggatatggttatcccaagtgattattttaagattgt aaaggaacataatcttcaagttttgggtctggtgaaaccagatgaagggttctatca gtgcattgctgaaaatgatgttggaaatgcacaagctggagcccaactgataatcct tgaacatgcaccagccacaacgggaccactgccttcagctcctcgggatgtcgtggc ctccctggtctctacccgcttcatcaaattgacgtggcggacacctgcatcagatcc tcacggagacaaccttacctactctgtgttctacaccaaggaagggattgctaggga acgtgttgagaataccagtcacccaggagagatgcaagtaaccattcaaaacctaat gccagcgaccgtgtacatctttagagttatggctcaaaataagcatggctcaggaga gagttcagctccactgcgagtagaaacacaacctgaggttcagctccctggcccagc acctaaccttcgtgcatatgcagcttcgcctacctccatcactgttacgtgggaaac accagtgtctggcaatggggaaattcagaattataaattgtactacatggaaaaggg gactgacaaagaacaggatgttgatgtttcaagtcacccttacaccattaatgggtt gaaaaaatatacagagtatagtttccgagtggtggcctacaataaacatggtcctgg agtttccacaccagatgttgctgttcgaacattgtcagatgctcccagtgctgctcc tcagaatctgtccttggaagtgagaaattcaaagagtattatgattcactggcagcc acctgctccagccacacaaaatgggcagattactggctacaagattcgctaccgaaa ggcctcccgaaagagtgatgtcactgagaccttggtaagcgggacacagctgtctca gctgattgaaggtcttgatcgggggactgagtataatttccgagtggctgctctaac aatcaatggtacaggcccggcaactgactggctgtctgctgaaacttttgaaagtga cctagatgaaactcgtgttcctgaagtgcctagctctcttcacgtacgcccgctcgt tactagcatcgtagtgagctggactcctccagagaatcagaacattgtggtcagagg ttacgccattggttatggcattggcagccctcatgcccagaccatcaaagtggacta taaacagcgctattacaccattgaaaatctggatcccagctctcactatgtgattac cctgaaagcatttaataacgtgggtgaaggcatccccctgtatgagagtgctgtgac caggcctcacacagacacttctgaagttgatttatttgttattaatgctccatacac tccagtgccagatcccactcccatgatgccaccagtgggagttcaggcttccattct gagtcatgacaccatcaggattacgtgggcagacaactcgctgcccaagcaccagaa gattacagactcccgatactacaccgtccgatggaaaaccaacatcccagcaaacac caagtacaagaatgcaaatgcaaccactttgagttatttggtgactggtttaaagcc gaatacactctatgaattctctgtgatggtgaccaaaggtcgaagatcaagtacatg gagtatgacagcccatgggaccacctttgaattagttccgacttctccacccaagga tgtgactgttgtgagtaaagaggggaaacctaagaccataattgtgaattggcagcc tccctccgaagccaatggcaaaattacaggttacatcatatattacagtacagatgt gaatgcagagatacatgactgggttattgagcctgttgtgggaaacagactgactca ccagatacaagagttaactcttgacacaccatactacttcaaaatccaggcacggaa ctcaaagggcatgggacccatgtctgaagctgtccaattcagaacacctaaagcgga ctcctctgataaaatgcctaatgatcaagcctcagggtctggagggaaaggaagccg gctgccagacctaggatccgactacaaacctccaatgagcggcagtaacagccctca tgggagccccacctctcctctggacagtaatatgctgctggtcataattgtttctgt tggcgtcatcaccatcgtggtggttgtgattatcgctgtcttttgtacccgtcgtac cacctctcaccagaaaaagaaacgagctgcctgcaaatcagtgaatggctctcataa gtacaaagggaattccaaagatgtgaaacctccagatctctggatccatcatgagag actggagctgaaacccattgataagtctccagacccaaaccccatcatgactgatac tccaattcctcgcaactctcaagatatcacaccagttgacaactccatggacagcaa tatccatcaaaggcgaaattcatacagagggcatgagtcagaggacagcatgtctac actggctggaaggcgaggaatgagaccaaaaatgatgatgccctttgactcccagcc accccagcctgtgattagtgcccatcccatccattccctcgacaaccctcaccatca tttccactccagcagcctcgcttctccagctcgcagtcatctctaccacccgggcag cccatggcccattggcacacccatgcccctttcagacagggccaattccacagaatc cgttcgaaatacccccagcactgacaccatgccagcctcttcgtctcaaacatgctg cactgatcaccaggaccctgaaggtgctaccagctcctcttacttggccagctccca agaggaagactcaggccagagtcttcccactgcccatgttcgcccttcccacccatt gaagagcttcgccgtgccagcaatcccgcctccaggacctcccacctatgatcctgc attgccaagcacaccattactgtcccagcaagctctgaaccatcacattcactcagt gaagacagcctccaccgggactctaggaaggagccggcctcctatgccagtggttgt tcccagtgcccctgaagtgcaggagaccacaaggatgttggaagactccgagagtag ctatgaaccagatgagctgaccaaagagatggcccacctggaaggactaatgaagga cctaaacgctatcacaacagcatgacgaccttcaccaggacctgacttcaaacctga gtctggaagtcttggaacttacccttgaaaacaaggaattgtacagagtacgagagg acagcacttgagaacacagaatgagccagcagactggccagcgcctctgtgtagggc tggctccaggcatggccacctgccttcccctggtcagcctggaagaagcctgtgtcg aggcagcttccctttgcctgctgatattctgcaggactgggcaccatgggccaaaat tttgtgtccagggaagaggcgagaagtgcaacctgcatttcactttgtggtcaggcc gtgtctttgtgctgtgactgcatcacctttatggagtgtagacattggcatttatgt acaattttatttgtgtcttattttattttaccttcaaaaacaaaaacgccatccaaa accaaggaagtccttggtgttctccacaagtggttgacatttgactgctcgttccaa ttatgtatggaaagtctttgacagcgtgggtcgttcctggggttggcttgttttttg gtttcatttttattttttaattctgagtcattgcatcctctaccagctgttaatcca tcactctgagggggaggaaatgttgcattgctgtttgtaagctttttttattatttt tttattataattattaaaggcctgactctttcctctcatcactgtgagattacagat ctatttgaattgaatgaaatgtaacattgaaaagacttgtttgttgctttctgtgca gtttcagtattggggcgggtggggggctgggggttggtaataggaaatggaggggct gctgaggtcctgtgaatgtttctgtcattgtactttcttccagaagcctgcagagaa tggaagcatcttctttattgtcctttcctggcatgtccatccttattgtcactacgt tgcaactggagtttgatttggatctggttttaaaattcttctgtgcaatagatgggt ttgaggatttagcggccctgatgtcttggtcatagcctggtaagaatgtccatgctg aggagccagatgttgtatttctaactgcctgagtcacacagaatagggtaagagcct gaccccattctgtaaatcagaaagcaaggatggagaccctttcctgctgctattatt ggctctctttgaggaagttggaggttaaggaaggaacttgtttgtttccgtatacga ctccttcttctctctagttcagtcttcagccagtccagcgctctcttccacacttca gagccccttcagagaaagcattagcaggaatgagacaaggcagagctgcagtgcccc ctgaggcttccacacatctttctgaatattatttttcaagtaacaagggcagggaca gcggaaacagctgcccaccccccccatcccagcagctcagctaagccctgatgagaa tgaagccacaggagttgtctgaggtgaacccagccgctcagccacacatggaagcca ttgcctttgcacatagttcttgggttctttttcctaaaaaggtaaggagctgaggtg tgtggttttttaatattaagaatatataatggaaaacacacgactgacgctcaggca tcttcccctactccccaacagatccccagaagacagcgtggaaggcagtgtagacag taaatcgggcttcagttctatagccaagaagagatcagctgctgaaaccaccagtgg gtaccccaggccacctgcctttgaacttggggatttgccatgtttgatcttgtcaca tacttgcttttttacaagatgaactctttgtatttatgatttggggggcaatgaaag gtgcaatgcaggaactgctgctgccgagctcgctggtcacatgggggtgccaggcgg gattctggaaaaccagtgcacttaaactgatcctgaagagagctgtcccagcactct ggccaccaggagggccagattccccagaaactaccttttgcccaaagaacatgctca gtatttggggcatttcctcccacaaaccctgactgcttctgttacctcagggccttg gtacctggatactgccacagaattggggcgggtgggggaggggcctatttttaaata aaataactgttcaaagttgggggttttttaaaaaattaagaaaaaggaaagctattc tgtattgcaccttttcacaatttaatacattttcttacattttcctgtgattttcga aactaaaccattgtgtgtcctgtagtgtcctggttgagctgccgctcagcagcttcc tcggggggatttggaacacctgtgtctgtcgtcgcactgcctgtgggaggggcccag agggctgctgggactggcgtctgtacacacttgtttggccttttctgtagttgatgc tgtaaactctatggctttttaaaaacgatttcatgtttttatttagtattggaaatc caatacacttttttaatccaatcaaaaaaaaaaaaaaaaaaaaaaa 29 Mouse NEO1- gcccccctcgctctaccgtgaagagcccgagtcggcgacgggtggcggcgcctggaa NM_001042752 cctggagagaccgagccaccccccggctctcggccggaatgtactgattctcctctg ctctcctccccgccccgctgcaggagggaggcgcccggagtctttccccctgggcgc gcgagggggccgcgcgggccgggccgggccgggctggagccgagccctgcggcgcag agaccggctgaggcgcgctgagggaagggcgcgagcgctccgcggcgctatcgccgc cgccgccgccgccactcgtggggtagagatggcggcggagcgcgaagccgggcgact cctctgcacctcctcctcccggcgctgctgcccgccaccgccgctgctgctgttgct gccgctgctgctgctgctcggacgcccggcgtccggcgccgcggccacgaagagcgg ctccccgccgcagtccgcaggagccagtgttcgaacattcactccgttttattttct ggtggagccagtagacaccctctcagttagaggctcttctgttatattaaattgctc ggcatattctgagccctctccaaacattgaatggaagaaagatgggacttttttaaa cttagaatcagatgatcgacgccagctactcccagatggatctttattcatcagcaa cgtggtgcattccaaacacaataagcctgacgaaggtttctatcagtgtgtagccac tgtggataatcttggaaccattgtcagcagaacagccaagctcacagtagcaggtct tccaagatttaccagccaaccagaaccttcttcagtctatgttggaaacagtgcaat tctgaattgtgaagttaatgcagatttggccccatttgttaggtgggaacagaatcg acagccccttcttctagatgacaggattgtcaaacttccaagtggaacactggttat cagcaatgctactgaaggagatgggggactctaccgctgcattgttgaaagtggtgg gccaccaaagtttagtgacgaagctgaattgaaagttcttcaagatcctgaggaaat tgtagacttggtatttctgatgcgaccatcttctatgatgaaagtcactggtcagag tgcagtgttgccatgtgttgtctcagggcttcctgctccagttgttagatggatgaa aaacgaagaagtgcttgacacagaaagctctggcaggttggtcttgctagcaggagg ttgcttggagatcagtgatgtcactgaggatgatgctgggacttatttttgcatagc tgataatggaaataagacagttgaagctcaggcggagcttactgtgcaagtgccacc tggattcctgaaacaacctgctaacatatatgctcacgaatccatagacattgtatt tgaatgtgaagtcactgggaagccaactccaactgtgaagtgggtcaagaatgggga tgtggttatccccagtgattactttaaaattgtaaaggaacataatcttcaagtttt gggtctggtgaaatcagatgaagggttctatcaatgcattgctgagaatgatgttgg aaatgcacaagctggagcccagctgataatccttgagcatgatgttgccatcccaac attacctcccacttcactgaccagtgccactactgaccatctagcaccagccacaac gggaccattaccttcagctcctcgagacgtcgtggcctccctggtctctactcgctt cattaaattgacatggcgtacacctgcatcagaccctcatggagacaatctcaccta ctctgtgttctacaccaaggaaggggttgctagggagcgtgttgagaataccagcca gccaggagagatgcaggtgactattcaaaacttgatgccagcaactgtgtacatctt caaagttatggctcaaaataagcatggctctggagaaagttcagctcctcttcgagt agagacacagcctgaggttcagctccctggcccagcacctaatatccgtgcttatgc aacgtcacctacttctatcactgtcacctgggaaacaccgttatctggcaatgggga aattcaaaattacaaattgtactacatggaaaaaggaactgataaagaacaggatat tgatgtttcaagtcactcctacaccattaatggactgaagaaatacacagaatacag tttccgagtggtggcctacaataaacatggtcctggagtttctacacaagatgttgc tgttcgaacattatcagatgttcccagtgctgctcctcagaatctgtccttagaagt gagaaattcaaagagtatagtgatccactggcagcccccttcctcaaccacacaaaa tgggcagataactggctacaagattcgatatcgaaaggcctcccgaaaaagtgatgt cactgagaccttggtaactgggacacagctgtctcagctgattgaaggtcttgatcg ggggacagaatataacttccgagtcgctgctctcacagtcaatggtacaggtccagc aactgattggctgtctgctgaaacttttgaaagcgacctagatgaaactcgtgttcc tgaagtgcccagctctcttcatgtccgtccgctcgtcactagcattgtagtgagctg gactcctccagagaaccagaacattgtggtccgaggttatgccatcggttacggcat tggcagccctcatgcccagaccatcaaagtggactataaacaacgttattacaccat cgaaaacttggatccaagctctcattacgtgattaccttgaaagcatttaacaatgt tggcgaaggcatccccctttatgagagtgctgtgaccagacctcacacagtgccaga tcccactcccatgatgccaccagtgggagttcaggcttccattctgagtcacgacac cataaggattacctgggcagacaactccctgcccaaacaccagaagattacagactc ccgctactacacagtccggtggaagaccaacatcccagcaaacacgaagtacaagaa tgcaaatgcaacgacgttaagctatttggttactggtttaaagccaaatacgctcta tgagttctctgtgatggtgaccaaaggcagaaggtcaagcacgtggagtatgacagc tcatggcgctacctttgaattagttcctacttctccacctaaggatgtgacagttgt gagtaaggaaggaaaacctagaaccatcatagtgaattggcagcctccctctgaagc taacggcaagattacaggttacatcatctattacagcacggatgtgaatgcagagat acatgactgggttattgaaccagttgtgggaaacagactgactcaccagattcaaga gttaacacttgatacgccatactacttcaaaatccaggcccggaactcaaaggacat ggggcccatgtctgaagctgtacagttcagaacacctaaagccttagggtcagcagg aaaaggaagccgactaccagacctgggatctgactacaaacctccaatgagtggcag caacagccctcacgggagccccacctcccctctggacagcaacatgctgctggtcat cattgtctctgttggcgtcatcactatcgtggtggttgtggtcattgctgctttttg tacccggcgcaccacctctcaccagaagaagaaacgagctgcgtgcaaatcagtgaa tggctcccataagtacaagggcaattgcaaagatgtgaagcctccagacctatggat ccatcacgagagactagagttgaagcctattgacaagtctccagatcctaaccctgt catgactgatactccaatcccctgaaactctcaagatatcacaccagtggacaattc catggatagcaatatccatcaaaggcggaattcatacagagggcatgagtcagagga cagcatgtctacactggctggaaggaggggaatgagaccaaaaatgatgatgccctt tgactctcagccacctcagcctgtgattagcgcccatcccatccattccctcgataa ccctcaccatcatttccactccagcagcctcgcttctccagcccgcagtcatctcta ccacccaagcagcccatggcccattggcacatccatgtccctttcagacagggccaa ttccacagaatctgttcgaaatacccccagcacggacaccatgccagcgtcctcgtc tcagacgtgctgcactgaccatcaggaccctgagggtgctactagctcctcttactt ggccagctcccaagaggaagactcaggccagagtcttcccacagcccatgtccgccc ttcccaccctctgaagagcttcgctgtgccagcaatcccacccccaggacctcctct ctatgatcctgcactgccaagcacaccattactgtcccagcaagctctgaaccatca cattcactcagtgaaaacagcctccatcgggacgttaggaaggagccggcctcctat gccagtggttgttccgagtgcccctgaagtacaggagaccaccaggatgctggaaga ctccgagagtagctatgaaccagatgagctgaccaaagagatggcccacctggaagg actaatgaaggacctaaatgccatcacaacagcctgatgaccttcgcctggacatga ctccaagcctgagtctacaagtctcggaacttaaccttgaaaacaaggaattgtaca gagtacgagaggacagcacttgagagcaggagccagcaaaccagccagtgcctccat gtggggttggctccaggcacagccacctgccttctcctggtcagcctggattacact tgtgtggaggcagcttccctttgcctgctgagagcctgcaggactgggcactatggg ccaaaattttgtgtccagggaagaggcaagaagtacgacctgccttttgctttgtgg tcagtggcttgtgtctttgtgctgcaactgcatcacttttatggagtgtagacattg gcatttatgtacaattttgtgtcctattttattttaccttaaaacactatcagaagc caagggagtctgtgatgttctctcaagcagttgacacttgactgtggttccagttac ttacggaaagtcatcaacagtgaggttgtttgacaccactgacaggcattggcttgt tgtgggtttcatttttattcttaattctgagacattgcatcctctgccagctgttaa tccatcactttgaggggaggacatgttgcattgctgtttgtaagcttttttattatt tttttattataattattaaaggcctgactttctcctctcatcactgtgagattacag atctatttgaatgaaatgtaacattgaaaagacttgtttgttgctttctgtgcagat tcagtattggggtgggattggggattgggaataggaaatggaggggctgctgaggcc ctgtgaatgtttctgtcaatgtactttgttccagaagcctgccgagaatgaaagcag catctttagtgtcctttcctggcatttccatcttcgtgtcaccgcatagcaactgga gttttgtttggatctggttataaattcttgtacagtggatgactttggtgatttagc tgccctggtatcttggtcatttcctctttggagtgtccacactgaggtctctatcaa tgtatgtttaattgcttgagagatgccaagtagaaccagagcctgactgtgctctga gaagctacaaagcacagggtggagactccctttgtgttgctagtattggttctctct ggaaggttaaaatctaaggcaggatcttggtttcctattccaaataggatgcctgct tctctgggcaccagtcctcagccaggcagctctcgtggcattgcagaggctctcctg aaaaacatcaaccagggtgagagccaagatggggtggcacccatgacgcttccccac atgtttcttcaaggagcagaggacagagatagtggaaagagggtcagcagaagcagg tgccttcatctatcccagcagctcagccaaaccccagttagaatgaggcagcaggag attccaggtgtgctgagggttcagccacacgcagaagacgttgcagagtgttaaaga ggtaagctgaggtgtgtatttggttggctttgttgttgttgttaatgtataatgaaa agtataagactaaccctcaggcctcatgttctccaatagatccctggaagacagtat agaaagtcagtcgggcttgggctccttagccagtgagactactcagaccaccagtgg ctagcctagcctacctgtccttgaacatgggtgattttacccctttgaggtcttaac cctttttttactttcaacaagatgagctctttgtatgattgcgggcgggggatatga aaatgcaatgatctaactcctgttgctcttctagctggtcacatgacggcaccaggc agggttctgggacacccggtgtgctttgactgttctacaaaaagctgccagagcgtt ctggcctcctggaggctagattcctcagaaactgtctagcctttgcccacagagcat gctatgtaattagagcactccttcccatgaaccccagcacttgtgttacctcagggc cttggtacctggatactgccacagaatttccatggggcgggaagggatgtattttta aataaagtaacttaaaagttggggaaattttttaaattcagaaaatgcaaagctatt ctgtattacaccatttcacaatttaatatgtcttatattttcctgtgactctggaaa ctaaaccattgtgtgtcttgtcgtgtcctagttgagctggggcctagcagcttcctt ccagtgggtgtggagcaaacgtgtatgtcgcctcgctacctgcttgaggggtccgaa gggctgctgggactgagttctgtacacacttgtttggccttttctgtagttgatgct gtaaaactctatggctttttaaaaacaatttcatgtttttattttgtattggaagtc caatacacttttttaatccaatcaaactggtctggtcaaaaagttctttcccttaaa agttcaggggctcctacttccagcttccgatgacttctctgtggctctcactgctat aaagcaggatttagaatggcaatctgggcagaggtaacaaaagaaatgtctgactgc cagccccaaaa

TABLE 2 siRNA targeting HAMP 3′ UTR SEQ SEQ Duplex name Start ID NO Sense (5′-3′) ID NO Antisense (5′-3′) 307-325_s 307 GGAUGUGCUGCAAGACGUA UACGUCUUGCAGCACAUCC 309-327_s 309 AUGUGCUGCAAGACGUAGA UCUACGUCUUGCAGCACAU 310-328_s 310 UGUGCUGCAAGACGUAGAA UUCUACGUCUUGCAGCACA 313-331_s 313 GCUGCAAGACGUAGAACCU AGGUUCUACGUCUUGUAGC AD-11439.1_31 314 CUGCAAGACGUAGAACCUA UAGGUUCUACGUCUUGCAG 4-332_s 322-340_s 322 CGUAGAACCUACCUGCCCU AGGGCAGGUAGGUUCUACG 347-365_s_G1A 347 GUCCCCUCCCUUCCUUAUU AAUAAGGAAGGGAGGGGAC 348-366_s 348 UCCCCUCCCUUCCUUAUUU AAAUAAGGAAGGGAGGGGA 349-367_s 349 CCCCUCCCUUCCUUAUUUA UAAAUAAGGAAGGGAGGGG 350-368_s 350 CCCUCCCUUCCUUAUUUAU AUAAAUAAGGAAGGGAGGG 351-369_s 351 CCUCCCUUCCUUAUUUAUU AAUAAAUAAGGAAGGGAGG 352-370_s_C19A 352 CUCCCUUCCUUAUUUAUUA UAAUAAAUAAGGAAGGGAG 352-370_s_C19U 352 CUCCCUUCCUUAUUUAUUU AAAUAAAUAAGGAAGGGAG 354-372_s 354 CCCUUCCUUAUUUAUUCCU AGGAAUAAAUAAGGAAGGG 355-373_s_G19A 355 CCUUCCUUAUUUAUUCCUA UAGGAAUAAAUAAGGAAGG 355-373_s_G19U 355 CCUUCCUUAUUUAUUCCUU AAGGAAUAAAUAAGGAAGG 356-374_s_C19A 356 CUUCCUUAUUUAUUCCUGA UCAGGAAUAAAUAAGGAAG 356-374_s_C19U 356 CUUCCUUAUUUAUUCCUGU ACAGGAAUAAAUAAGGAAG 357-375_s 357 UUCCUUAUUUAUUCCUGCU AGCAGGAAUAAAUAAGGAA 358-376_s_G19A 358 UCCUUAUUUAUUCCUGCUA UAGCAGGAAUAAAUAAGGA 358-376_s_G19U 358 UCCUUAUUUAUUCCUGCUU AAGCAGGAAUAAAUAAGGA 359-377_s_C19A 359 CCUUAUUUAUUCCUGCUGA UCAGCAGGAAUAAAUAAGG 359-377_s_C19U 359 CCUUAUUUAUUCCUGCUGU ACAGCAGGAAUAAAUAAGG 363-381_s 363 AUUUAUUCCUGCUGCCCCA UGGGGCAGCAGGAAUAAAU 365-383_s 365 UUAUUCCUGCUGCCCCAGA UCUGGGGCAGCAGGAAUAA 366-384_s 366 UAUUCCUGCUGCCCCAGAA UUCUGGGGCAGCAGGAAUA 369-387_s 369 UCCUGCUGCCCCAGAACAU AUGUUCUGGGGCAGCAGGA 370-388_s 370 CCUGCUGCCCCAGAACAUA UAUGUUCUGGGGCAGCAGG 373-391_s 373 GCUGCCCCAGAACAUAGGU ACCUAUGUUCUGGGGCAGC 375-393_s 375 UGCCCCAGAACAUAGGUCU AGACCUAUGUUCUGGGGCA 376-394_s 376 GCCCCAGAACAUAGGUCUU AAGACCUAUGUUCUGGGGC 379-397_s 379 CCAGAACAUAGGUCUUGGA UCCAAGACCUAUGUUCUGG 380-398_s 380 CAGAACAUAGGUCUUGGAA UUCCAAGACCUAUGUUCUG 381-399_s 381 AGAACAUAGGUCUUGGAAU AUUCCAAGACCUAUGUUCU AD-11442.1_38 382 GAACAUAGGUCUUGGAAUA UAUUCCAAGACCUAUGUUC 2-400_s 383-401_s 383 AACAUAGGUCUUGGAAUAA UUAUUCCAAGACCUAUGUU 396-414_s 396 GAAUAAAAUGGCUGGUUCU AGAACCAGCCAUUUUAUUC 398-416_s 398 AUAAAAUGGCUGGUUCUUU AAAGAACCAGCCAUUUUAU 399-417_s 399 UAAAAUGGCUGGUUCUUUU AAAAGAACCAGCCAUUUUA 402-420_s 402 AAUGGCUGGUUCUUUUGUU AACAAAAGAACCAGCCAUU 403-421_s 403 AUGGCUGGUUCUUUUGUUU AAACAAAAGAACCAGCCAU 407-425_s 407 CUGGUUCUUUUGUUUUCCA UGGAAAACAAAAGAACCAG AD-11436.1_29 291 CAUCGAUCAAAGUGUGGGA UCCCACACUUUGAUCGAUG 1-309_s Note that an overhang (e.g. TT, dTsdT) can be added to the 3′ end of any duplex.

TABLE 3 siRNA targeting HAMP CDS SEQ SEQ Duplex name Start ID NO sense (5′-3′) ID NO Antisense (5′-3′) 62-80_s_G19U  62 AGACGGCACGAUGGCACUU AAGUGCCAUCGUGCCGUCU 67-85_s_C19A  67 GCACGAUGGCACUGAGCUA UAGCUCAGUGCCAUCGUGC 67-85_s_C19U  67 GCACGAUGGCACUGAGCUU AAGCUCAGUGCCAUCGUGC 74-92_s_C19A  74 GGCACUGAGCUCCCAGAUA UAUCUGGGAGCUCAGUGCC 74-92_s_C19U  74 GGCACUGAGCUCCCAGAUU AAUCUGGGAGCUCAGUGCC 76-94_s_G19A  76 CACUGAGCUCCCAGAUCUA UAGAUCUGGGAGCUCAGUG 76-94_s_G19U  76 CACUGAGCUCCCAGAUCUU AAGAUCUGGGAGCUCAGUG 132-150_s 132 CUGACCAGUGGCUCUGUUU AAACAGAGCCACUGGUCAG 140-158_s 140 UGGCUCUGUUUUCCCACAA UUGUGGGAAAACAGAGCCA 146-164_s_hcU1C_G19A 146 UGUUUUCCCACAACAGACA UGUCUGUUGUGGGAAAACA 146-164_s_hcU1C_G19U 146 UGUUUUCCCACAACAGACU AGUCUGUUGUGGGAAAACA 155-173_s 155 ACAACAGACGGGACAACUU AAGUUGUCCCGUCUGUUGU 157-175_s_C19A 157 AACAGACGGGACAACUUGA UCAAGUUGUCCCGUCUGUU 157-175_s_C19U 157 AGACGGGACAACUUGCAGA UCUGCAAGUUGUCCCGUCU 160-178_s 160 AGACGGGACAACUUGCAGA UCUGCAAGUUGUCCCGUCU 161-179_s_G19A 161 GACGGGACAACUUGCAGAA UUCUGCAAGUUGUCCCGUC 161-179_s_G19U 161 GACGGGACAACUUGCAGAU AUCUGCAAGUUGUCCCGUC 162-180_s_C19A 162 ACGGGACAACUUGCAGAGA UCUCUGCAAGUUGUCCCGU 162-180_s_C19U 162 ACGGGACAACUUGCAGAGU ACUCUGCAAGUUGUCCCGU 242-260_s_C19A 242 GAGGCGAGACACCCACUUA UAAGUGGGUGUCUCGCCUC 242-260_s_C19U 242 GAGGCGAGACACCCACUUU AAAGUGGGUGUCUCGCCUC 253-271_s 253 CCCACUUCCCCAUCUGCAU AUGCAGAUGGGGAAGUGGG 258-276_s 258 UUCCCCAUCUGCAUUUUCU AGAAAAUGCAGAUGGGGAA 261-279_s 261 CCCAUCUGCAUUUUCUGCU AGCAGAAAAUGCAGAUGGG 275-293_s 275 CUGCUGCGGCUGCUGUCAU AUGACAGCAGCCGCAGCAG 276-294_s_C19A 276 UGCUGCGGCUGCUGUCAUA UAUGACAGCAGCCGCAGCA 276-294_s_C19U 276 UGCUGCGGCUGCUGUCAUU AAUGACAGCAGCCGCAGCA 278-296_s 278 CUGCGGCUGCUGUCAUCGA UCGAUGACAGCAGCCGCAG 279-297_s 279 UGCGGCUGCUGUCAUCGAU AUCGAUGACAGCAGCCGCA 280-298_s_C19A 280 GCGGCUGCUGUCAUCGAUA UAUCGAUGACAGCAGCCGC 280-298_s_C19U 280 GCGGCUGCUGUCAUCGAUU AAUCGAUGACAGCAGCCGC 281-299_s 281 CGGCUGCUGUCAUCGAUCA UGAUCGAUGACAGCAGCCG AD-11443.1_282-300_s 282 GGCUGCUGUCAUCGAUCAA UUGAUCGAUGACAGCAGCC AD-11432.1_283-301_s 283 GCUGCUGUCAUCGAUCAAA UUUGAUCGAUGACAGCAGC 284-302_s_G19A 284 CUGCUGUCAUCGAUCAAAA UUUUGAUCGAUGACAGCAG 284-302_s_G19U 284 CUGCUGUCAUCGAUCAAAU AUUUGAUCGAUGACAGCAG AD-11441.1_285-303_s 285 UGCUGUCAUCGAUCAAAGU ACUUUGAUCGAUGACAGCA 286-304_s_G19A 286 GCUGUCAUCGAUCAAAGUA UACUUUGAUCGAUGACAGC 286-304_s_G19U 286 GCUGUCAUCGAUCAAAGUU AACUUUGAUCGAUGACAGC AD-11447.1_297-305_s 287 CUGUCAUCGAUCAAAGUGU ACACUUUGAUCGAUGACAG 288-306_s_G19A 288 UGUCAUCGAUCAAAGUGUA UACACUUUGAUCGAUGACA 288-306_s_G19U 288 UGUCAUCGAUCAAAGUGUU AACACUUUGAUCGAUGACA 290-308_s_G19A 290 UCAUCGAUCAAAGUGUGGA UCCACACUUUGAUCGAUGA 290-308_s_G19U 290 UCAUCGAUCAAAGUGUGGU ACCACACUUUGAUCGAUGA 295-313_s_G19A 295 GAUCAAAGUGUGGGAUGUA UACAUCCCACACUUUGAUC 295-313_s_G19U 295 GAUCAAAGUGUGGGAUGUU AACAUCCCACACUUUGAUC 299-317_s_C19U 299 AAAGUGUGGGAUGUGCUGU ACAGCACAUCCCACACUUU Note that an overhang (e.g. TT, dTsdT) can be added to the 3′ end of any duplex.

Table 4: HAMP Modified Sequences

TABLE 4 SEQ Antisense SEQ Start Sense ID ID Target Duplex ID Position Name Sense Sequence NO Name Antisense Sequence NO HAMP AD-45073   2 A-94166.1 AcuGucAcucGGucccAGA A-94167.1 UCUGGGACCGAGUGAcAGU dTsdT dTsdT HAMP AD-45079   7 A-94168.1 cAcucGGucccAGAcAccA A-94169.1 UGGUGUCUGGGACCGAGUG dTsdT dTsdT HAMP AD-45085  16 A-94170.1 ccAGAcAccAGAGcAAGcu A-94171.1 AGCUUGCUCUGGUGUCUGG dTsdT dTsdT HAMP AD-29928  43 A-66808.1 AGcAGuGGGAcAGccAGAc A-66809.1 GUCUGGCUGUCCcACUGCU dTsdT dTsdT HAMP AD-45674  43 A-95618.1 AGcAGuGGGAcAGccAGAA A-95619.1 UUCUGGCUGUCCcACUGCU dTsdT dTsdT HAMP AD-45680  43 A-95620.1 AGcAGuGGGAcAGccAGAu A-95621.1 AUCUGGCUGUCCcACUGCU dTsdT dTsdT HAMP AD-45686  48 A-95622.1 uGGGAcAGccAGAcAGAcG A-95623.1 CGUCUGUCUGGCUGUCCcA dTsdT dTsdT HAMP AD-45698  48 A-95626.1 uGGGAcAGccAGAcAGAcu A-95627.1 AGUCUGUCUGGCUGUCCcA dTsdT dTsdT HAMP AD-45692  48 A-95624.1 uGGGAcAGccAGAcAGAcA A-95625.1 UGUCUGUCUGGCUGUCCcA dTsdT dTsdT HAMP AD-45354  51 A-94701.1 GAcAGccAGAcAGAcGGcA A-94702.1 UGCCGUCUGUCUGGCUGUC dTsdT dTsdT HAMP AD-29929  54 A-66810.1 AGccAGAcAGAcGGcAcGA A-66811.1 UCGUGCCGUCUGUCUGGCU dTsdT dTsdT HAMP AD-45091  55 A-94172.1 GccAGAcAGAcGGcAcGAu A-94173.1 AUCGUGCCGUCUGUCUGGC dTsdT dTsdT HAMP AD-29930  59 A-66812.1 GAcAGAcGGcAcGAuGGcA A-66813.1 UGCcAUCGUGCCGUCUGUC dTsdT dTsdT HAMP AD-29931  60 A-66814.1 AcAGAcGGcAcGAuGGcAc A-66815.1 GUGCcAUCGUGCCGUCUGU dTsdT dTsdT HAMP AD-45704  60 A-95628.1 AcAGAcGGcAcGAuGGcAA A-95629.1 UUGCcAUCGUGCCGUCUGU dTsdT dTsdT HAMP AD-45710  60 A-95630.1 AcAGAcGGcAcGAuGGcAu A-95631.1 AUGCcAUCGUGCCGUCUGU dTsdT dTsdT HAMP AD-29932  61 A-66816.1 cAGAcGGcAcGAuGGcAcu A-66817.1 AGUGCcAUCGUGCCGUCUG dTsdT dTsdT HAMP AD-47030  62 A-98344.1 AGACfGGCfACfGAUfGGC A-98345.1 AAGUGCCfAUCGUGCCGUC fACfUfUfdTsdT UdTsdT HAMP AD-29933  62 A-66818.1 AGAcGGcAcGAuGGcAcuG A-66819.1 cAGUGCcAUCGUGCCGUCU dTsdT dTsdT HAMP AD-45675  62 A-95634.1 AGAcGGcAcGAuGGcAcuu A-95635.1 AAGUGCcAUCGUGCCGUCU dTsdT dTsdT HAMP AD-45716  62 A-95632.1 AGAcGGcAcGAuGGcAcuA A-95633.1 uAGUGCcAUCGUGCCGUCU dTsdT dTsdT HAMP AD-29934  63 A-66820.1 GAcGGcAcGAuGGcAcuGA A-66821.1 UcAGUGCcAUCGUGCCGUC dTsdT dTsdT HAMP AD-29935  64 A-66822.1 AcGGcAcGAuGGcAcuGAG A-66823.1 CUcAGUGCcAUCGUGCCGU dTsdT dTsdT HAMP AD-45687  64 A-95638.1 AcGGcAcGAuGGcAcuGAu A-95639.1 AUcAGUGCcAUCGUGCCGU dTsdT dTsdT HAMP AD-45681  64 A-95636.1 AcGGcAcGAuGGcAcuGAA A-95637.1 UUcAGUGCcAUCGUGCCGU dTsdT dTsdT HAMP AD-29936  66 A-66824.1 GGcAcGAuGGcAcuGAGcu A-66825.1 AGCUcAGUGCcAUCGUGCC dTsdT dTsdT HAMP AD-47043  67 A-98348.1 GCfACfGAUfGGCfACfUf A-98349.1 AAGCUCfAGUGCCfAUCGU GAGCfUfUfdTsdT GCdTsdT HAMP AD-47037  67 A-98346.1 GCfACfGAUfGGCfACfUf A-98347.1 CfAGCUCfAGUGCCfAUCG GAGCfUfAdTsdT UGCdTsdT HAMP AD-29937  67 A-66826.1 GcAcGAuGGcAcuGAGcuc A-66827.1 GAGCUcAGUGCcAUCGUGC dTsdT dTsdT HAMP AD-45699  67 A-95642.1 GcAcGAuGGcAcuGAGcuu A-95643.1 AAGCUcAGUGCcAUCGUGC dTsdT dTsdT HAMP AD-45693  67 A-95640.1 GcAcGAuGGcAcuGAGcuA A-95641.1 uAGCUcAGUGCcAUCGUGC dTsdT dTsdT HAMP AD-45711  68 A-95646.1 cAcGAuGGcAcuGAGcucA A-95647.1 UGAGCUcAGUGCcAUCGUG dTsdT dTsdT HAMP AD-45717  68 A-95648.1 cAcGAuGGcAcuGAGcucu A-95649.1 AGAGCUcAGUGCcAUCGUG dTsdT dTsdT HAMP AD-45705  68 A-95644.1 cAcGAuGGcAcuGAGcucc A-95645.1 GGAGCUcAGUGCcAUCGUG dTsdT dTsdT HAMP AD-45682  69 A-95652.1 AcGAuGGcAcuGAGcuccA A-95653.1 UGGAGCUcAGUGCcAUCGU dTsdT dTsdT HAMP AD-45688  69 A-95654.1 AcGAuGGcAcuGAGcuccu A-95655.1 AGGAGCUcAGUGCcAUCGU dTsdT dTsdT HAMP AD-45676  69 A-95650.1 AcGAuGGcAcuGAGcuccc A-95651.1 GGGAGCUcAGUGCcAUCGU dTsdT dTsdT HAMP AD-45360  70 A-94703.1 cGAuGGcAcuGAGcucccA A-94704.1 UGGGAGCUcAGUGCcAUCG dTsdT dTsdT HAMP AD-45366  71 A-94705.1 GAuGGcAcuGAGcucccAG A-94706.1 CUGGGAGCUcAGUGCcAUC dTsdT dTsdT HAMP AD-29938  72 A-66828.1 AuGGcAcuGAGcucccAGA A-66829.1 UCUGGGAGCUcAGUGCcAU dTsdT dTsdT HAMP AD-45372  73 A-94707.1 uGGcAcuGAGcucccAGAu A-94708.1 AUCUGGGAGCUcAGUGCcA dTsdT dTsdT HAMP AD-47055  74 A-98352.1 GGCfACfUfGAGCfUfCfC A-98353.1 AAUCUGGGAGCUCfAGUGC fCfAGAUfUfdTsdT CdTsdT HAMP AD-47049  74 A-98350.1 GGCfACfUfGAGCfUfCfC A-98351.1 CfAUCUGGGAGCUCfAGUG fCfAGAUfAdTsdT CCdTsdT HAMP AD-45700  74 A-95658.1 GGcAcuGAGcucccAGAuu A-95659.1 AAUCUGGGAGCUcAGUGCC dTsdT dTsdT HAMP AD-29939  74 A-66830.1 GGcAcuGAGcucccAGAuc A-66831.1 GAUCUGGGAGCUcAGUGCC dTsdT dTsdT HAMP AD-45694  74 A-95656.1 GGcAcuGAGcucccAGAuA A-95657.1 uAUCUGGGAGCUcAGUGCC dTsdT dTsdT HAMP AD-29940  75 A-66832.1 GcAcuGAGcucccAGAucu A-66833.1 AGAUCUGGGAGCUcAGUGC dTsdT dTsdT HAMP AD-47067  76 A-98356.1 CfACfUfGAGCfUfCfCfC A-98357.1 AAGAUCUGGGAGCUCfAGU fAGAUfCfUfUfdTsdT GdTsdT HAMP AD-47061  76 A-98354.1 CfACfUfGAGCfUfCfCfC A-98355.1 CfAGAUCUGGGAGCUCfAG fAGAUfCfUfAdTsdT UGdTsdT HAMP AD-45712  76 A-95662.1 cAcuGAGcucccAGAucuu A-95663.1 AAGAUCUGGGAGCUcAGUG dTsdT dTsdT HAMP AD-29941  76 A-66834.1 cAcuGAGcucccAGAucuG A-66835.1 cAGAUCUGGGAGCUcAGUG dTsdT dTsdT HAMP AD-45706  76 A-95660.1 cAcuGAGcucccAGAucuA A-95661.1 uAGAUCUGGGAGCUcAGUG dTsdT dTsdT HAMP AD-45097  88 A-94174.1 AGAucuGGGccGcuuGccu A-94175.1 AGGcAAGCGGCCcAGAUCU dTsdT dTsdT HAMP AD-45103  91 A-94176.1 ucuGGGccGcuuGccuccu A-94177.1 AGGAGGcAAGCGGCCcAGA dTsdT dTsdT HAMP AD-45378 116 A-94709.1 ccuccuccucGccAGccuG A-94710.1 cAGGCUGGCGAGGAGGAGG dTsdT dTsdT HAMP AD-45383 117 A-94711.1 cuccuccucGccAGccuGA A-94712.1 UcAGGCUGGCGAGGAGGAG dTsdT dTsdT HAMP AD-45388 118 A-94713.1 uccuccucGccAGccuGAc A-94714.1 GUcAGGCUGGCGAGGAGGA dTsdT dTsdT HAMP AD-45393 120 A-94715.1 cuccucGccAGccuGAccA A-94716.1 UGGUcAGGCUGGCGAGGAG dTsdT dTsdT HAMP AD-45355 121 A-94717.1 uccucGccAGccuGAccAG A-94718.1 CUGGUcAGGCUGGCGAGGA dTsdT dTsdT HAMP AD-45361 122 A-94719.1 ccucGccAGccuGAccAGu A-94720.1 ACUGGUcAGGCUGGCGAGG dTsdT dTsdT HAMP AD-45367 123 A-94721.1 cucGccAGccuGAaccAGuG A-94722.1 cACUGGUcAGGCUGGCGAG dTsdT dTsdT HAMP AD-45373 126 A-94723.1 GccAGccuGAccAGuGGcu A-94724.1 AGCcACUGGUcAGGCUGGC dTsdT dTsdT HAMP AD-45109 132 A-94178.1 cuGAccAGuGGcucuGuuu A-94179.1 AAAcAGAGCcACUGGUcAG dTsdT dTsdT HAMP AD-47032 140 A-98360.1 UfGGCfUfCfUfGUfUfUf A-98361.1 UUGUGGGAAAACfAGAGCC UfCfCfCfACfAAdTsdT fAdTsdT HAMP AD-45115 140 A-94180.1 uGGcucuGuuuucccAcAA A-94181.1 UUGUGGGAAAAcAGAGCcA dTsdT dTsdT HAMP AD-45074 142 A-94182.1 GcucuGuuuucccAcAAcA A-94183.1 UGUUGUGGGAAAAcAGAGC dTsdT dTsdT HAMP AD-47038 146 A-98362.1 UfGUfUfUfUfCfCfCfAC A-98363.1 UGUCUGUUGUGGGAAAACf fAACfAGACfAdTsdT AdTsdT HAMP AD-47044 146 A-98364.1 UfGUfUfUfUfCfCfCfAC A-98365.1 AGUCUGUUGUGGGAAAACf fAACfAGACfUfdTsdT AdTsdT HAMP AD-45677 146 A-95666.1 uGuuuucccAcAAcAGAcA A-95667.1 UGUCUGUUGUGGGAAAAcA dTsdT dTsdT HAMP AD-45683 146 A-95668.1 uGuuuucccAcAAcAGAcu A-95669.1 AGUCUGUUGUGGGAAAAcA dTsdT dTsdT HAMP AD-45718 146 A-95664.1 uGuuuucccAcAAcAGAcG A-95665.1 CGUCUGUUGUGGGAAAAcA dTsdT dTsdT HAMP AD-45080 149 A-94184.1 uuucccAcAAcAGAcGGGA A-94185.1 UCCCGUCUGUUGUGGGAAA dTsdT dTsdT HAMP AD-45379 150 A-94725.1 uucccAcAAcAGAcGGGAc A-94726.1 GUCCCGUCUGUUGUGGGAA dTsdT dTsdT HAMP AD-29942 151 A-66836.1 ucccAcAAcAGAcGGGAcA A-66837.1 UGUCCCGUCUGUUGUGGGA dTsdT dTsdT HAMP AD-29943 152 A-66838.1 cccAcAAcAGAcGGGAcAA A-66839.1 UUGUCCCGUCUGUUGUGGG dTsdT dTsdT HAMP AD-29944 153 A-66840.1 ccAcAAcAGAcGGGAcAAc A-15142.2 GUUGUCCCGUCUGUUGUGG dTsdT dTsdT HAMP AD-45695 153 A-95672.1 ccAcAAcAGAcGGGAcAAu A-95673.1 AUUGUCCCGUCUGUUGUGG dTsdT dTsdT HAMP AD-45689 153 A-95670.1 ccAcAAcAGAcGGGAcAAA A-95671.1 UUUGUCCCGUCUGUUGUGG dTsdT dTsdT HAMP AD-29945 154 A-66841.1 cAcAAcAGAcGGGAcAAcu A-15116.1 AGUUGUCCCGUCUGUUGUG dTsdT dTsdT HAMP AD-47050 155 A-98366.1 ACfAACfAGACfGGGACfA A-15182.3 AAGUUGUCCCGUCUGUUGU ACfUfUfdTsdT dTsdT HAMP AD-29946 155 A-66842.1 AcAAcAGAcGGGAcAAcuu A-15182.1 AAGUUGUCCCGUCUGUUGU dTsdT dTsdT HAMP AD-47062 157 A-98369.1 AACfAGACfGGGACfAACf A-98370.1 ACfAAGUUGUCCCGUCUGU UfUfGUfdTsdT UdTsdT HAMP AD-47056 157 A-98367.1 AACfAGACfGGGACfAACf A-98368.1 UCfAAGUUGUCCCGUCUGU UfUfGAdTsdT UdTsdT HAMP AD-45713 157 A-95678.1 AAcAGAcGGGAcAAcuuGu A-95679.1 AcAAGUUGUCCCGUCUGUU dTsdT dTsdT HAMP AD-45707 157 A-95676.1 AAcAGAcGGGAcAAcuuGA A-95677.1 UcAAGUUGUCCCGUCUGUU dTsdT dTsdT HAMP AD-45701 157 A-95674.1 AAcAGAcGGGAcAAcuuGc A-95675.1 GcAAGUUGUCCCGUCUGUU dTsdT dTsdT HAMP AD-45394 159 A-94727.1 cAGAcGGGAcAAcuuGcAG A-94728.1 CUGcAAGUUGUCCCGUCUG dTsdT dTsdT HAMP AD-47068 160 A-98371.1 AGACfGGGACfAACfUfUf A-98372.1 UCUGCfAAGUUGUCCCGUC GCfAGAdTsdT UdTsdT HAMP AD-45389 160 A-94729.1 AGAcGGGAcAAcuuGcAGA A-94730.1 UCUGcAAGUUGUCCCGUCU dTsdT dTsdT HAMP AD-47033 161 A-98375.1 GACfGGGACfAACfUfUfG A-98376.1 AUCUGCfAAGUUGUCCCGU CfAGAUfdTsdT CdTsdT HAMP AD-47074 161 A-98373.1 GACfGGGACfAACfUfUfG A-98374.1 UUCUGCfAAGUUGUCCCGU CfAGAAdTsdT CdTsdT HAMP AD-45678 161 A-95682.1 GAcGGGAcAAcuuGcAGAu A-95683.1 AUCUGcAAGUUGUCCCGUC dTsdT dTsdT HAMP AD-45719 161 A-95680.1 GAcGGGAcAAcuuGcAGAA A-95681.1 UUCUGcAAGUUGUCCCGUC dTsdT dTsdT HAMP AD-29947 161 A-66843.1 GAcGGGAcAAcuuGcAGAG A-66844.1 CUCUGcAAGUUGUCCCGUC dTsdT dTsdT HAMP AD-47039 162 A-98377.1 ACfGGGACfAACfUfUfGC A-98378.1 UCUCUGCfAAGUUGUCCCG fAGAGAdTsdT UdTsdT HAMP AD-47045 162 A-98379.1 ACfGGGACfAACfUfUfGC A-98380.1 ACUCUGCfAAGUUGUCCCG fAGAGUfdTsdT UdTsdT HAMP AD-45690 162 A-95686.1 AcGGGAcAAcuuGcAGAGA A-95687.1 UCUCUGcAAGUUGUCCCGU dTsdT dTsdT HAMP AD-45696 162 A-95688.1 AcGGGAcAAcuuGcAGAGu A-95689.1 ACUCUGcAAGUUGUCCCGU dTsdT dTsdT HAMP AD-45684 162 A-95684.1 AcGGGAcAAcuuGcAGAGc A-95685.1 GCUCUGcAAGUUGUCCCGU dTsdT dTsdT HAMP AD-30016 163 A-66845.1 cGGGAcAAcuuGcAGAGcu A-66846.1 AGCUCUGcAAGUUGUCCCG dTsdT dTsdT HAMP AD-45394 164 A-94731.1 GGGAcAAcuuGcAGAGcuG A-94732.1 cAGCUCUGcAAGUUGUCCC dTsdT dTsdT HAMP AD-45702 165 A-95690.1 GGAcAAcuuGcAGAGcuGc A-95691.1 GcAGCUCUGcAAGUUGUCC dTsdT dTsdT HAMP AD-45708 165 A-95692.1 GGAcAAcuuGcAGAGcUGA A-95693.1 UcAGCUCUGcAAGUUGUCC dTsdT dTsdT HAMP AD-45714 165 A-95694.1 GGAcAAcuuGcAGAGcuGu A-95695.1 AcAGCUCUGcAAGUUGUCC dTsdT dTsdT HAMP AD-29949 166 A-66847.1 GAcAAcuuGcAGAGcuGcA A-66848.1 UGcAGCUCUGcAAGUUGUC dTsdT dTsdT HAMP AD-45086 167 A-94186.1 AcAAcuuGcAGAGcuGcAA A-94187.1 UUGcAGCUCUGcAAGUUGU dTsdT dTsdT HAMP AD-45356 168 A-94733.1 cAAcuuGcAGAGcuGcAAc A-94734.1 GUUGcAGCUCUGcAAGUUG dTsdT dTsdT HAMP AD-45685 169 A-95700.1 AAcuuGcAGAGcuGcAAcu A-95701.1 AGUUGcAGCUCUGcAAGUU dTsdT dTsdT HAMP AD-45679 169 A-95698.1 AAcuuGcAGAGcuGcAAcA A-95699.1 UGUUGcAGCUCUGcAAGUU dTsdT dTsdT HAMP AD-45720 169 A-95696.1 AAcuuGcAGAGcuGcAAcc A-95697.1 GGUUGcAGCUCUGcAAGUU dTsdT dTsdT HAMP AD-45703 170 A-95706.1 AcuuGcAGAGcuGcAAccu A-95707.1 AGGUUGcAGCUCUGcAAGU dTsdT dTsdT HAMP AD-45697 170 A-95704.1 AcuuGcAGAGcuGcAAccA A-95705.1 UGGUUGcAGCUCUGcAAGU dTsdT dTsdT HAMP AD-45691 170 A-95702.1 AcuuGcAGAGcuGcAAccc A-95703.1 GGGUUGcAGCUCUGcAAGU dTsdT dTsdT HAMP AD-45362 189 A-94735.1 cAGGAcAGAGcuGGAGccA A-94736.1 UGGCUCcAGCUCUGUCCUG dTsdT dTsdT HAMP AD-45368 190 A-94737.1 AGGAcAGAGcuGGAGccAG A-94738.1 CUGGCUCcAGCUCUGUCCU dTsdT dTsdT HAMP AD-45374 199 A-94739.1 cuGGAGccAGGGccAGcuG A-94740.1 cAGCUGGCCCUGGCUCcAG dTsdT dTsdT HAMP AD-45092 222 A-94188.1 cccAuGuuccAGAGGcGAA A-94189.1 UUCGCCUCUGGAAcAUGGG dTsdT dTsdT HAMP AD-45721 228 A-95712.1 uuccAGAGGcGAAGGAGGu A-95713.1 ACCUCCUUCGCCUCUGGAA dTsdT dTsdT HAMP AD-45715 228 A-95710.1 uuccAGAGGcGAAGGAGGA A-95711.1 UCCUCCUUCGCCUCUGGAA dTsdT dTsdT HAMP AD-45709 228 A-95708.1 uuccAGAGGcGAAGGAGGc A-95709.1 GCCUCCUUCGCCUCUGGAA dTsdT dTsdT HAMP AD-45380 230 A-94741.1 ccAGAGGcGAAGGAGGcGA A-94742.1 UCGCCUCCUUCGCCUCUGG dTsdT dTsdT HAMP AD-45385 231 A-94743.1 cAGAGGcGAAGGAGGcGAG A-94744.1 CUCGCCUCCUUCGCCUCUG dTsdT dTsdT HAMP AD-29950 232 A-66849.1 AGAGGcGAAGGAGGcGAGA A-66850.1 UCUCGCCUCCUUCGCCUCU dTsdT dTsdT HAMP AD-45390 233 A-94745.1 GAGGcGAAGGAGGcGAGAc A-94746.1 GUCUCGCCUCCUUCGCCUC dTsdT dTsdT HAMP AD-29951 234 A-66851.1 AGGcGAAGGAGGcGAGAcA A-66852.1 UGUCUCGCCUCCUUCGCCU dTsdT dTsdT HAMP AD-45395 235 A-94747.1 GGcGAAGGAGGcGAGAcAc A-94748.1 GUGUCUCGCCUCCUUCGCC dTsdT dTsdT HAMP AD-45727 239 A-95714.1 AAGGAGGcGAGAcAcccAA A-95715.1 UUGGGUGUCUCGCCUCCUU dTsdT dTsdT HAMP AD-45732 239 A-95716.1 AAGGAGGcGAGAcAcccAu A-95717.1 AUGGGUGUCUCGCCUCCUU dTsdT dTsdT HAMP AD-29952 239 A-66853.1 AAGGAGGcGAGAcAcccAc A-66854.1 GUGGGUGUCUCGCCUCCUU dTsdT dTsdT HAMP AD-29953 240 A-66855.1 AGGAGGcGAGAcAcccAcu A-66856.1 AGUGGGUGUCUCGCCUCCU dTsdT dTsdT HAMP AD-30017 241 A-66857.1 GGAGGcGAGAcAcccAcuu A-66858.1 AAGUGGGUGUCUCGCCUCC dTsdT dTsdT HAMP AD-47057 242 A-98383.1 GAGGCfGAGACfACfCfCf A-95721.2 AAAGUGGGUGUCUCGCCUC ACfUfUfUfdTsdT dTsdT HAMP AD-47051 242 A-98381.1 GAGGCfGAGACfACfCfCf A-98382.1 CfAAGUGGGUGUCUCGCCU ACfUfUfAdTsdT CdTsdT HAMP AD-30018 242 A-66859.1 GAGGcGAGAcAcccAcuuc A-66860.1 GAAGUGGGUGUCUCGCCUC dTsdT dTsdT HAMP AD-45737 242 A-95718.1 GAGGcGAGAcAcccAcuuA A-95719.1 uAAGUGGGUGUCUCGCCUC dTsdT dTsdT HAMP AD-29956 246 A-66861.1 cGAGAcAcccAcuuccccA A-66862.1 UGGGGAAGUGGGUGUCUCG dTsdT dTsdT HAMP AD-45357 247 A-94749.1 GAGAcAcccAcuuccccAu A-94750.1 AUGGGGAAGUGGGUGUCUC dTsdT dTsdT HAMP AD-45363 248 A-94751.1 AGAcAcccAcuuccccAuc A-94752.1 GAUGGGGAAGUGGGUGUCU dTsdT dTsdT HAMP AD-45747 251 A-95722.1 cAcccAcuuccccAucuGc A-95723.1 GcAGAUGGGGAAGUGGGUG dTsdT dTsdT HAMP AD-45752 251 A-95724.1 cAcccAcuuccccAucuGA A-95725.1 UcAGAUGGGAAGUGGGUG dTsdT dTsdT HAMP AD-45757 251 A-95726.1 cAcccAcuuccccAucuGu A-95727.1 AcAGAUGGGGAAGUGGGUG dTsdT dTsdT HAMP AD-29957 252 A-66863.1 AcccAcuuccccAucuGcA A-66864.1 UGcAGAUGGGGAAGUGGGU dTsdT dTsdT HAMP AD-47063 253 A-98384.1 CfCfCfACfUfUfCfCfCf A-98385.1 AUGCfAGAUGGGGAAGUGG CfAUfCfUfGCfAUfdTsdT GdTsdT HAMP AD-45399 253 A-94753.1 cccAcuuccccAucuGcAu A-94754.1 AUGcAGAUGGGGAAGUGGG dTsdT dTsdT HAMP AD-45098 255 A-94190.1 cAcuuccccAucuGcAuuu A-94191.1 AAAUGcAGAUGGGGAAGUG dTsdT dTsdT HAMP AD-45400 256 A-94755.1 AcuuccccAucuGcAuuuu A-94756.1 AAAAUGcAGAUGGGGAAGU dTsdT dTsdT HAMP AD-45381 257 A-94757.1 cuuccccAucuGcAuuuuc A-94758.1 GAAAAUGcAGAUGGGGAAG dTsdT dTsdT HAMP AD-47069 258 A-98386.1 UfUfCfCfCfCfAUfCfUf A-98387.1 AGAAAAUGCfAGAUGGGGA GCfAUfUfUfUfCfUfdTs AdTsdT dT HAMP AD-45401 258 A-94759.1 uuccccAucuGcAuuuucu A-94760.1 AGAAAAUGcAGAUGGGGAA dTsdT dTsdT HAMP AD-47075 261 A-98388.1 CfCfCfAUfCfUfGCfAUf A-98389.1 AGCfAGAAAAUGCfAGAUG UfUfUfCfUfGCfUfdTsdT GGdTsdT HAMP AD-29958 261 A-66865.1 cccAucuGcAuuuucuGcu A-66866.1 AGcAGAAAAUGcAGAUGGG dTsdT dTsdT HAMP AD-45391 262 A-94761.1 ccAucuGcAuuuucuGcuG A-94762.1 cAGcAGAAAAUGcAGAUGG dTsdT dTsdT HAMP AD-29959 267 A-66867.1 uGcAuuuucuGcuGcGGcu A-66868.1 AGCCGcAGcAGAAAAUGcA dTsdT dTsdT HAMP AD-29960 268 A-66869.1 GcAuuuucuGcuGcGGcuG A-66870.1 cAGCCGcAGcAGAAAAUGC dTsdT dTsdT HAMP AD-30019 270 A-66871.1 AuuuucuGcuGcGGcuGcu A-66872.1 AGcAGCCGcAGcAGAAAAU dTsdT dTsdT HAMP AD-45396 271 A-94763.1 uuuucuGcuGcGGcuGcuG A-94764.1 cAGcAGCCGcAGcAGAAAA dTsdT dTsdT HAMP AD-45358 272 A-94765.1 uuucuGcuGcGGcuGcuGu A-94766.1 AcAGcAGCCGcAGcAGAAA dTsdT dTsdT HAMP AD-45364 273 A-94767.1 uucuGcuGcGGcuGcuGuc A-94768.1 GAcAGcAGCCGcAGcAGAA dTsdT dTsdT HAMP AD-29962 274 A-66873.1 ucuGcuGcGGcuGcuGucA A-66874.1 UGAcAGcAGCCGcAGcAGA dTsdT dTsdT HAMP AD-47034 275 A-98390.1 CfUfGCfUfGCfGGCfUfG A-98391.1 AUGACfFGCfAGCCGCfAG CfUfGUfCfAUfdTsdT CfAGdTsdT HAMP AD-45370 275 A-94769.1 cuGcuGcGGcuGcuGucAu A-94770.1 AUGAcAGcAGCCGcAGcAG dTsdT dTsdT HAMP AD-47046 276 A-98394.1 UfGCfUfGCfGGCfUfGCf A-98395.1 AAUGACfAGCfAGCCGCfA UfGUfCfAUfUfdTsdT GCfAdTsdT HAMP AD-47040 276 A-98392.1 UfGCfUfGCfGGCfUfGCf A-98393.1 CfAUGACfAGCfAGCCGCf UfGUfCfAUfAdTsdT AGCfAdTsdT HAMP AD-45728 276 A-95730.1 uGcuGcGGcuGcuGucAuu A-95731.1 AAUGAcAGcAGCCGcAGcA dTsdT dTsdT HAMP AD-45722 276 A-95728.1 uGcuGcGGcuGcuGucAuA A-95729.1 uAUGAcAGcAGCCGcAGcA dTsdT dTsdT HAMP AD-29963 276 A-66875.1 uGcuGcGGcuGcuGucAuc A-66876.1 GAUGAcAGcAGCCGcAGcA dTsdT dTsdT HAMP AD-45104 278 A-94192.1 cuGcGGcuGcuGucAucGA A-94193.1 UCGAUGAcAGcAGCCGcAG dTsdT dTsdT HAMP AD-47058 279 A-98398.1 UfGCfGGCfUfGCfUfGUf A-98399.1 AUCGAUGACfAGCfAGCCG CfAUfCfGAUfdTsdT CfAdTsdT HAMP AD-29964 279 A-66877.1 uGcGGcuGcuGucAucGAu A-66878.1 AUCGAUGAcAGcAGCCGcA dTsdT dTsdT HAMP AD-47070 280 A-98402.1 GCfGGCfUfGCfUfGUfCf A-98403.1 AAUCGAUGACfAGCfAGCC AUfCfGAUfUfdTsdT GCdTsdT HAMP AD-47064 280 A-98400.1 GCfGGCfUfGCfUfGUfCf A-98401.1 CfAUCGAUGACfAGCfAGC AUfCfGAUfFdTsdT CGCdTsdT HAMP AD-45738 280 A-95734.1 GcGGcuGcuGucAucGAuu A-95735.1 AAUCGAUGAcAGcAGCCGC dTsdT dTsdT HAMP AD-45733 280 A-95732.1 GcGGcuGcuGucAucGAuA A-95733.1 uAUCGAUGAcAGcAGCCGC dTsdT dTsdT HAMP AD-47076 281 A-98404.1 CfGGCfUfGCfUfGUfCfA A-98405.1 UGAUCGAUGACfAGCfAGC UfCfGAUfCfAdTsdT CGdTsdT HAMP AD-29965 281 A-66879.1 cGGcuGcuGucAucGAucA A-66880.1 UGAUCGAUGAcAGcAGCCG dTsdT dTsdT HAMP AD-47035 282 A-98406.1 GGCfUfGCfUfGUfCfAUf A-98407.1 UUGAUCGAUGACfAGCfAG CfGAUfCfAAdTsdT CCdTsdT HAMP AD-47041 283 A-98408.1 GCfUfGCfUfGUfCfAUfC A-98409.1 UUUGAUCGAUGACfAGCfA fGAUfCfAAAdTsdT GCdTsdT HAMP AD-30020 283 A-18260.1 GcuGcuGucAucGAucAAA A-18261.1 UUUGAUCGAUGAcAGcAGC dTsdT dTsdT HAMP AD-47053 284 A-98412.1 CfUfGCfUfGUfCfAUfCf A-98413.1 AUUUGAUCGAUGACfAGCf GAUfCfAAAUfdTsdT AGdTsdT HAMP AD-47047 284 A-98410.1 CfUfGCfUfGUfCfAUfCf A-98411.1 UUUUGAUCGAUGACfAGCf GAUfCfAAAAdTsdT AGdTsdT HAMP AD-45748 284 A-95738.1 cuGcuGucAucGAucAAAu A-95739.1 AUUUGAUCGAUGAcAGcAG dTsdT dTsdT HAMP AD-45743 284 A-95736.1 cuGcuGucAucGAucAAAA A-95737.1 UUUUGAUCGAUGAcAGcAG dTsdT dTsdT HAMP AD-30021 284 A-18284.1 cuGcuGucAucGAucAAAG A-18285.1 CUUUGAUCGAUGAcAGcAG dTsdT dTsdT HAMP AD-47059 285 A-98414.1 UfGCfUfGUfCfAUfCfGA A-98415.1 ACUUUGAUCGAUGACfAGC UfCfAAAGUfdTsdT fAdTsdT HAMP AD-11441 285 A-18278.3 uGcuGucAucGAucAAAGu A-18279.2 ACUUUGAUCGAUGAcAGcA dTsdT dTsdT HAMP AD-47071 286 A-98418.1 GCfUfGUfCfAUfCfGAUf A-98419.1 AACUUUGAUCGAUGACfA CfAAAGUfUfdTsdT GCdTsdT HAMP AD-47065 286 A-98416.1 GCfUfGUfCfAUfCfGAUf A-98417.1 CfACUUUGAUCGAUGACfA CfAAAGUfAdTsdT GCdTsdT HAMP AD-45758 286 A-95742.1 GcuGucAcuGAcuAAAGuu A-95743.1 AACUUUGAUCGAUGAcAGC dTsdT dTsdT HAMP AD-45753 286 A-95740.1 GcuGucAucGAucAAAGuA A-95741.1 uACUUUGAUCGAUGAcAGC dTsdT dTsdT HAMP AD-29968 286 A-18288.1 GcuGucAucGAucAAAGuG A-18289.1 cACUUUGAUCGAUGAcAGC dTsdT dTsdT HAMP AD-47077 287 A-98420.1 CfUfGUfCfAUfCfGAUfC A-98421.1 ACfACUUUGAUCGAUGACf fAAAGUfGUfdTsdT AGdTsdT HAMP AD-29969 287 A-18290.3 cuGucAucGAucAAAgUgU A-18291.1 AcACUUUGAUCGAUGAcAG dTsdT dTsdT HAMP AD-48208 288 A-100241.2 uGucAucGAucAAAGuGuu A-100243.1 AACACUUUgAuCgAuGaCa dTsdT dTsdT HAMP AD-47042 288 A-98424.1 UfGUfCfAUfCfFAUfCfA A-98425.1 AACfACUUUGAUCGAUGAC AAGUfGUfUfdTsdT fAdTsdT HAMP AD-48202 288 A-100241.1 uGucAcuGAucAAAGuGuu A-100242.1 AACACuuUGauCGAuGaca dTsdT dTsdT HAMP AD-47036 288 A-98422.1 UfGUfCfAUfCfGAUfCfA A-98423.1 CfACfACUUUGAUCGAUGA AAGUfGUfAdTsdT CfAdTsdT HAMP AD-45729 288 A-95746.1 uGucAucGAucAAAGuGuu A-95747.1 AAcACUUUGAUCGAUGAcA dTsdT dTsdT HAMP AD-45723 288 A-95744.1 uGucAucGAucAAAGuGuA A-95745.1 uAcACUUUGAUCGAUGAcA dTsdT dTsdT HAMP AD-29970 288 A-66881.1 uGucAucGAucAAAGuGuG A-66882.1 cAcACUUUGAUCGAUGAcA dTsdT dTsdT HAMP AD-47048 290 A-98426.1 UfCfAUfCfGAUfCfAAAG A-98427.1 UUCfACfACUUUGAUCGAU UfGUfGGAdTsdT GAdTsdT HAMP AD-47054 290 A-98428.1 UfCfAUfCfGAUfCfAAAG A-98429.1 ACCfACfACUUUGAUCGAU UfGUfGGUfdTsdT GAdTsdT HAMP AD-45744 290 A-95752.1 ucAucGAucAAAGuGuGGu A-95753.1 ACcAcACUUUGAUCGAUGA dTsdT dTsdT HAMP AD-45739 290 A-95750.1 ucAucGAucAAAGuGuGGA A-95751.1 UCcAcACUUUGAUCGAUGA dTsdT dTsdT HAMP AD-45734 290 A-95748.1 ucAucGAucAAAGuGuGGG A-95749.1 CCcAcACUUUGAUCGAUGA dTsdT dTsdT HAMP AD-47005 291 A-98342.1 CfAUfCfGAUfCfAAAGUf A-98343.1 UCCCfACfACUUUGAUCGA GUfGGGAdTsdT UGdTsdT HAMP AD-11436 291 A-18268.1 cAucGAucAAAGuGuGGGA A-18269.1 UCCcAcACUUUGAUCGAUG dTsdT dTsdT HAMP AD-11436 291 A-18268.1 cAucGAucAAAGuGuGGGA A-18269.1 UCCcAcACUUUGAUCGAUG dTsdT dTsdT HAMP AD-29971 291 A-18268.1 cAucGAucAAAGuGuGGGA A-18269.1 UCCcAcACUUUGAUCGAUG dTsdT dTsdT HAMP AD-45376 292 A-94771.1 AucGAucAAAGuGuGGGAu A-94772.1 AUCCcAcACUUUGAUCGAU dTsdT dTsdT HAMP AD-45382 293 A-94773.1 ucGAucAAAGuGuGGGAuG A-94774.1 cAUCCcAcACUUUGAUCGA dTsdT dTsdT HAMP AD-29972 294 A-66883.1 cGAucAAAGuGuGGGAuGu A-66884.1 AcAUCCcAcACUUUGAUCG dTsdT dTsdT HAMP AD-47066 295 A-98432.1 GAUfCfAAAGUfGUfGGGA A-98433.1 AACfAUCCCfACfACUUUG UfFUfUfdTsdT AUdTsdT HAMP AD-47060 295 A-98430.1 GAUfCfAAAGUfGUfGGGA A-98431.1 CfACfAUCCCfACfACUUU UfGUfAdTsdT GAUCdTsdT HAMP AD-45754 295 A-95756.1 GAucAAAGuGuGGGAuGuu A-95757.1 AAcAUCCcAcACUUUGAUC dTsdT dTsdT HAMP AD-45749 295 A-95754.1 GAucAAAGuGuGGGAuGuA A-95755.1 uAcAUCCcAcACUUUGAUC dTsdT dTsdT HAMP AD-29973 295 A-66885.1 GAucAAAGuGuGGGAuGuG A-66886.1 cAcAUCCcAcACUUUGAUC dTsdT dTsdT HAMP AD-45730 296 A-95762.1 AucAAAGuGuGGGAuGuGu A-95763.1 AcAcAUCCcAcACUUUGAU dTsdT dTsdT HAMP AD-45724 296 A-95760.1 AucAAAGuGuGGGAuGuGA A-95761.1 UcAcAUCCcAcACUUUGAU dTsdT dTsdT HAMP AD-45759 296 A-95758.1 AucAAAGuGuGGGAuGuGc A-95759.1 GcAcAUCCcAcACUUUGAU dTsdT dTsdT HAMP AD-45110 297 A-94194.1 ucAAAGuGuGGGAuGuGcu A-94195.1 AGcAcAUCCcAcACUUUGA dTsdT dTsdT HAMP AD-45387 298 A-94775.1 cAAAGuGuGGGAuGuGcuG A-94776.1 cAGcAcAUCCcAcACUUUG dTsdT dTsdT HAMP AD-47072 299 A-98434.1 AAAGUfGUfGGGAUfGUfG A-98435.1 ACfAGCfACfAUCCCfACf CfUfGUfdTsdT ACUUUdTsdT HAMP AD-45740 299 A-95766.1 AAAGuGuGGGAuGuGcuGA A-95767.1 UcAGcAcAUCCcAcACUUU dTsdT dTsdT HAMP AD-45745 299 A-95768.1 AAAGuGuGGGAuGuGcuGu A-95769.1 AcAGcAcAUCCcAcACUUU dTsdT dTsdT HAMP AD-45735 299 A-95764.1 AAAGuGuGGGAuGuGcuGc A-95765.1 GcAGcAcAUCCcAcACUUU dTsdT dTsdT HAMP AD-29974 300 A-66887.1 AAGuGuGGGAuGuGcuGcA A-66888.1 UGcAGcAcAUCCcAcACUU dTsdT dTsdT HAMP AD-29975 301 A-66889.1 AGuGuGGGAuGuGcuGcAA A-66890.1 UUGcAGcAcAUCCcAcACU dTsdT dTsdT HAMP AD-45116 306 A-94196.1 GGGAuGuGcuGcAAGAcGu A-94197.1 ACGUCUUGcAGcAcAUCCC dTsdT dTsdT HAMP AD-46988 307 A-98258.1 GGAUfGUfGCfUfGCfAAG A-98259.1 CfACGUCUUGCfAGCfACf ACfGUfAdTsdT AUCCdTsdT HAMP AD-45075 307 A-94198.1 GGAuGuGcuGcAAGAcGuA A-94199.1 uACGUCUUGcAGcAcAUCC dTsdT dTsdT HAMP AD-46994 309 A-98260.1 AUfGUfGCfUfGCfAAGAC A-98261.1 UCCfACGUCUUGCfAGCfA fGUfAGAdTsdT CfAUdTsdT HAMP AD-45081 309 A-94200.1 AuGuGcuGcAAGAcGuAGA A-94201.1 UCuACGUCUUGcAGcAcAU dTsdT dTsdT HAMP AD-47000 310 A-98262.1 UfGUfGCfUfGCfAAGACf A-98263.1 UUCCfACGUCUUGCfAGCf GUfAGAAdTsdT ACfAdTsdT HAMP AD-45087 310 A-94202.1 uGuGcuGcAAGAcGuAGAA A-94203.1 UUCuACGUCUUGcAGcAcA dTsdT dTsdT HAMP AD-47006 313 A-98264.1 GCfUfGCfAAGACfGUfAG A-98265.1 AGGUUCCfACGUCUUGCfA AACfCfUfdTsdT GCdTsdT HAMP AD-45093 313 A-94204.1 GcuGcAAGAcGuAGAAccu A-94205.1 AGGUUCuACGUCUUGcAGC dTsdT dTsdT HAMP AD-47011 314 A-98266.1 CfUfGCfAAGACfGUfAGA A-98267.1 CfAGGUUCCfACGUCUUGC ACfCfUfAdTsdT fAGdTsdT HAMP AD-47016 322 A-98268.1 CfGUfAGAACfCfUfACfC A-98269.1 AGGGCfAAGCfAGGUUCCf fUfGCfCfCfUfdTsdT ACGdTsdT HAMP AD-45099 322 A-94206.1 cGuAGAAccuAccuGcccu A-94207.1 AGGGcAGGuAGGUUCuACG dTsdT dTsdT HAMP AD-47021 347 A-98270.1 GUfCfCfCfCfUfCfCfCf A-98271.1 AACfAAGGAAGGGAGGGGA UfUfCfCfUfUfAUfUfdT CdTsdT sdT HAMP AD-47026 348 A-98272.1 UfCfCfCfCfUfCfCfCfU A-98273.1 AAACfAAGGAAGGGAGGGG fUfCfCfUfUfAUfUfdTs AdTsdT dT HAMP AD-46989 349 A-98274.1 CfCfCfCfUfCfCfCfUfU A-98275.1 CfAAACfAAGGAAGGGAGG FCfCfUfUfAUfUfUfAdT GGdTsdT sdT HAMP AD-46995 350 A-98276.1 CfCfCfUfCfCfCfUfUfC A-98277.1 ACfAAACfAAGGAAGGGAG fCfUfUfAUfUfUfAUfdT GGdTsdT sdT HAMP AD-47001 351 A-98278.1 CfCfUfCfCfCfUfUfCfC A-98279.1 AACfAAACfAAGGAAGGGA fUfUfAUfUfUfAUfUfdT GGdTsdT sdT HAMP AD-47012 352 A-98282.1 CfUfCfCfCfUfUfCfCfU A-98283.1 AAACfAAACfAAGGAAGGG fUfAUfUfUfAUfUfUfdT AGdTsdT sdT HAMP AD-47007 352 A-98280.1 CfUfCfCfCfUfUfCfCfU A-98281.1 CfAACfAAACfAAGGAAGG fUfAUfUfUfAUfUfAdTs GAGdTsdT dT HAMP AD-47017 354 A-98284.1 CfCfCfUfUfCfCfUfUfA A-98285.1 AGGAACfAAACfAAGGAAG UfUfUfAUfUfCfCfUfdT GGdTsdT sdT HAMP AD-47022 355 A-98286.1 CfCfUfUfCfCfUfUfAUf A-98287.1 CfAGGAACfAAACfAAGGA UfUfAUfUfCfCfUfAdTs AGGdTsdT dT HAMP AD-47027 355 A-98288.1 CfCfUfUfCfCfUfUfAUf A-98289.1 AAGGAACfAAACfAAGGAA UfUfAUfUfCfCfUfUfdT GGdTsdT sdT HAMP AD-46996 356 A-98292.1 CfUfUfCfCfUfUfAUfUf A-98293.1 ACfAGGAACfAAACfAAGG UfAUfUfCfCfUfGUfdTs AAGdTsdT dT HAMP AD-46990 356 A-98290.1 CfUfUfCfCfUfUfAUfUf A-98291.1 UCfAGGAACfAAACfAAGG UfAUfUfCfCfUfGAdTs AAGdTsdT dT HAMP AD-47002 357 A-98294.1 UfUfCfCfUfUfAUfUfUf A-98295.1 AGCfAGGAACfAAACfAAG AUfUfCfCfUfGCfUfdT GAAdTsdT sdT HAMP AD-47013 358 A-98298.1 UfCfCfUfUfAUfUfUfAU A-98299.1 AAGCfAGGAACfAAACfAA fUfCfCfUfGCfUfUfdTs GGAdTsdT dT HAMP AD-47008 358 A-98296.1 UfCfCfUfUfAUfUfUfAU A-98297.1 CfAGCfAGGAACfAAACfA fUfCfCfUfGCfUfAdTs AGGAdTsdT dT HAMP AD-47023 359 A-98302.1 CfCfUfUfAUfUfUfAUfU A-98303.1 ACfAGCfAGGAACfAAACf fCfCfUfGCfUfGUfdTs AAGGdTsdT dT HAMP AD-47018 359 A-98300.1 CfCfUfUfAUfUfUfAUfU A-98301.1 UCfAGCfAGGAACfAAACf UCfCfUfGCfUfGAdTs AAGGdTsdT dT HAMP AD-47028 363 A-98304.1 AUfUfUfAUfUfCfCfCfG A-98305.1 UGGGGCfAGCfAGGAACfA CfUfGCfCfCfCfAdTsdT AAUdTsdT HAMP AD-46991 365 A-98306.1 UfUfAUfUfCfCfUfGCfU A-98307.1 UCUGGGGCfAGCfAGGAAC fGCfCfCfCfAGAdTsdT fAAdTsdT HAMP AD-46997 366 A-98308.1 UfAUfUfCfCfUfGCfUfG A-98309.1 UUCUGGGGCfAGCfAGGAA CfCfCfCfAGAAdTsdT CfAdTsdT HAMP AD-47003 369 A-98310.1 UfCfCfUfGCfUfGCfCfC A-98311.1 AUGUUCUGGGGCfAGCfAG fCfAGAACfAUfdTsdT GAdTsdT HAMP AD-45105 369 A-94208.1 uccuGcuGccccAGAAcAu A-94209.1 AUGUUCUGGGGcAGcAGGA dTsdT dTsdT HAMP AD-47009 370 A-98312.1 CfCfUfGCfUfGCfCfCfC A-98313.1 CfAUGUUCUGGGGCfAGCf fAGAACfAUfAdTsdT AGGdTsdT HAMP AD-45111 370 A-94210.1 ccuGcuGccccAGAAcAuA A-94211.1 uAUGUUCUGGGGcAGcAGG dTsdT dTsdT HAMP AD-47014 373 A-98314.1 GCfUfGCfCfCfCfAGAAC A-98315.1 ACCCfAUGUUCUGGGGCfA fAUfAGGUfdTsdT GCdTsdT HAMP AD-45117 373 A-94212.1 GcuGccccAGAAcAuAGGu A-94213.1 ACCuAUGUUCUGGGGcAGC dTsdT dTsdT HAMP AD-47019 375 A-98316.1 UfGCfCfCfCfAGAACfAU A-98317.1 AGACCCfAUGUUCUGGGGC fAGGUfCfUfdTsdT fAdTsdT HAMP AD-45076 375 A-94214.1 uGccccAGAAcAuAGGucu A-94215.1 AGACCuAUGUUCUGGGGcA dTsdT dTsdT HAMP AD-48214 376 A-100244.1 GccccAGAAcAuAGGucuu A-100245.1 AAGACCuAUGUUCUGGGGC dTdT dTdT HAMP AD-48219 376 A-100246.1 GcCCCAGAAcAuAGGucuu A-100247.1 AAGACCuaUGuuCUGGGGc dTdT dTdT HAMP AD-47024 376 A-98318.1 GCfCfCfCfAGAACfAUfA A-98319.1 AAGACCCfAUGUUCUGGGG GGUfCfUfUfdTsdT CdTsdT HAMP AD-45082 376 A-94216.1 GccccAGAAcAuAGGucuu A-94217.1 AAGACCuAUGUUCUGGGGC dTsdT dTsdT HAMP AD-48224 379 A-100248.1 ccAGAAcAuAGGucuuGGA A-100249.1 UCcAAGACCuAUGUUCUCG dTdT dTdT HAMP AD-48187 379 A-100248.2 ccAGAAcAuAGGucuuGGA A-100250.1 uCCAAGACCuAUGuUCugg dTdT dTdT HAMP AD-47029 379 A-98320.1 CfCfAGAACfAUfAGGUfC A-98321.1 UCCfAAGACCCfAUGUUCU fUfUfGGAdTsdT GGdTsdT HAMP AD-48192 379 A-100248.3 ccAGAAcAuAGGucuuGGA A-100251.1 uCCAAGACCUaUgUuCuGg dTdT dTdT HAMP AD-45088 379 A-94218.1 ccAGAAcAuAGGucuuGGA A-94219.1 UCcAAGACCuAUGUUCUGG dTsdT dTsdT HAMP AD-46992 380 A-98322.1 CfAGAACfAUfAGGUfCfU A-98323.1 UUCCfAAGACCCfAUGUUC fUfGGAAdTsdT UGdTsdT HAMP AD-45094 380 A-94220.1 cAGAAcAuAGGucuuGGAA A-94221.1 UUCcAAGACCuAUGUUCUG dTsdT dTsdT HAMP AD-46998 381 A-98324.1 AGAACfAUfAGGUfCfUfU A-98325.1 AUUCCfAAGACCCfAUGUU fGGAAUfdTsdT CUdTsdT HAMP AD-45100 381 A-94222.1 AGAAcAuAGGucuuGGAAu A-94223.1 AUUCcAAGACCuAUGUUCU dTsdT dTsdT HAMP AD-48137 382 A-100195.1 GAAcAuAGGUCUUGGAAUA A-98136.7 UAUUCCAAGACCUAUGUUC dTdT dTdT HAMP AD-48196 382 A-100179.22 GAAcAuAGGucuuGGAAuA A-100228.1 UAuUCCAAGaCCuAuGuuc dTdT dTdT HAMP AD-48195 382 A-98135.8 GAACAUAGGUCUUGGAAUA A-100218.1 UAUUcCaAgAcCuAuGuUc dTdT dTdT HAMP AD-48201 382 A-100179.23 GAAcAuAGGucuuGGAAuA A-100229.1 UAUUCCAAGaCCuAuGuuc dTdT dTdT HAMP AD-48207 382 A-100179.24 GAAcAuAGGucuuGGAAuA A-100230.1 UAUUCCAAgAcCuAuGuuc dTdT dTdT HAMP AD-48159 382 A-100179.5 GAAcAuAGGucuuGGAAuA A-100184.1 UAUUCCAAGACCUAuGuUC dTdT dTdT HAMP AD-48147 382 A-100179.3 GAAcAuAGGucuuGGAAuA A-100182.1 UAUUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48161 382 A-98135.4 GAACAUAGGUCUUGGAAUA A-100188.2 UAUUCCAAGACCUAuGuUc dTdT dTdT HAMP AD-48172 382 A-100193.1 GAAcAuAGGucUUGGAAUA A-98136.5 UAUUCCAAGACCUAUGUUC dTdT dTdT HAMP AD-48156 382 A-100194.4 GAAcAuAGGuCUUGGAAUA A-100188.3 UAUUCCAAGACCUAuGuUc dTdT dTdT HAMP AD-48195 382 A-98135.8 GAACAUAGGUCUUGGAAUA A-100218.1 UAUUcCaAgAcCuAuGuUc dTdT dTdT HAMP AD-48136 382 A-100179.9 GAAcAuAGGucuuGGAAuA A-100187.1 UAUUCCAAGACCUAUGuUc dTdT dTdT HAMP AD-48166 382 A-100192.1 GAAcAuAGGucuUGGAAUA A-98136.1 UAUUCCAAGACCUAUGUUC dTdT dTdT HAMP AD-48213 382 A-100179.25 GAAcAuAGGucuuGGAAuA A-100231.1 UAuUCCAAgAcCuAuGuuc dTdT dTdT HAMP AD-48173 382 A-98135.6 GAACAUAGGUCUUGGAAUA A-100190.2 UAUUCCAAGACcuAuGuUc dTdT dTdT HAMP AD-48154 382 A-100179.12 GAAcAuAGGucuuGGAAuA A-100190.1 UAUUCCAAGACcuAuGuUc dTdT dTdT HAMP AD-48141 382 A-100179.2 GAAcAuAGGuCUUGGAAUA A-100181.1 UAuUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48216 382 A-100217.3 GAAcAuAGGuCUUGGAAUA A-100215.3 UAUUCCAAGACcuAuGuUc dTsdT dTsdT HAMP AD-48180 382 A-100194.8 GAAcAuAGGuCUUGGAAUA A-100183.2 UAUUCCAAGACCuAuGuUC dTdT dTdT HAMP AD-48143 382 A-100196.1 GAAcAUAGGUCUUGGAAUA A-98136.8 UAUUCCAAGACCUAUGUUC dTdT dTdT HAMP AD-48142 382 A-100179.10 GAAcAuAGGucuuGGAAuA A-100188.1 UAUUCCAAGACCUAuGuUc dTdT dTdT HAMP AD-48221 382 A-18280.13 GAAcAuAGGucuuGGAAuA A-15168.3 UAUUCCAAGACCUAUGUUC dTsdT dTsdT HAMP AD-48171 382 A-100179.7 GAAcAUAGGucuuGGAAuA A-98136.2 UAUUCCAAGACCUAUGUUC dTdT dTdT HAMP AD-48145 382 A-100195.4 GAAcAuAGGUCUUGGAAUA A-100183.3 UAUUCCAAGACCuAuGuUC dTdT dTdT HAMP AD-48160 382 A-100191.1 GAAcAuAGGucuuGGAAUA A-98136.3 UAUUCCAAGACCUAUGUUC dTdT dTdT HAMP AD-48144 382 A-100194.2 GAAcAuAGGuCUUGGAAUA A-100190.5 UAUUCCAAGACcuAuGuUc dTdT dTdT HAMP AD-48167 382 A-98135.5 GAACAUAGGUCUUGGAAUA A-100189.2 UAUUCCAAGACCuAuGuUc dTdT dTdT HAMP AD-48177 382 A-100179.8 GAAcAuAGGucuuGGAAuA A-100186.1 UAUUCCAAGACCUAUGUUc dTdT dTdT HAMP AD-48153 382 A-100179.4 GAAcAuAGGucuuGGAAuA A-100183.1 UAUUCCAAGACCuAuGuUC dTdT dTdT HAMP AD-48178 382 A-100194.1 GAAcAuAGGuCUUGGAAUA A-98136.6 UAUUCCAAGACCUAUGUUC dTdT dTdT HAMP AD-48155 382 A-98135.3 GAACAUAGGUCUUGGAAUA A-100187.2 UAUUCCAAGACCUAUGuUc dTdT dTdT HAMP AD-48174 382 A-100194.7 GAAcAuAGGuCUUGGAAUA A-100197.1 UAUUCCAAGACcuAuGuUC dTdT dTdT HAMP AD-48205 382 A-15167.2 GAACAUAGGUCUUGGAAUA A-100215.2 UAUUCCAAGACcuAuGuUc dTsdT dTsdT HAMP AD-48179 382 A-100196.2 GAAcAUAGGUCUUGGAAUA A-100190.3 UAUUCCAAGACcuAuGuUc dTdT dTdT HAMP AD-48168 382 A-100194.6 GAAcAuAGGuCUUGGAAUA A-100186.3 UAUUCCAAGACCUAUGUUc dTdT dTdT HAMP AD-48149 382 A-98135.2 GAACAUAGGUCUUGGAAUA A-100186.2 UAUUCCAAGACCUAUGUUc dTdT dTdT HAMP AD-48211 382 A-100217.2 GAAcAuAGGuCUUGGAAUA A-100214.2 UAUUCCAAGACCuAuGuUC dTsdT dTsdT HAMP AD-48200 382 A-100217.1 GAAcAuAGGuCUUGGAAUA A-15168.2 UAUUCCAAGACCUAUGUUC dTsdT dTsdT HAMP AD-48188 382 A-100179.20 GAAcAuAGGucuuGGAAuA A-100205.1 UAuUCcAAGACCuAuGuUc dTdT dTdT HAMP AD-48183 382 A-18280.10 GAAcAuAGGucuuGGAAuA A-100214.1 UAUUCCAAGACCuAuGuUC dTsdT dTsdT HAMP AD-48150 382 A-100194.3 GAAcAuAGGuCUUGGAAUA A-100189.3 UAUUCCAAGACCuAuGuUc dTdT dTdT HAMP AD-48162 382 A-100194.5 GAAcAuAGGuCUUGGAAUA A-100187.3 UAUUCCAAGACCUAUGuUc dTdT dTdT HAMP AD-48139 382 A-100195.3 GAAcAuAGGUCUUGGAAUA A-100197.2 UAUUCCAAGACcuAuGuUC dTdT dTdT HAMP AD-9940 382 A-15167.1 GAACAUAGGUCUUGGAAUA A-15168.2 UAUUCCAAGACCUAUGUUC dTsdT dTsdT HAMP AD-48138 382 A-100195.2 GAAcAuAGGUCUUGGAAUA A-100190.4 UAUUCCAAGACcuAuGuUc dTdT dTdT HAMP AD-11459 382 A-18280.2 GAAcAuAGGucuuGGAAuA A-18304.1 uAuUCcAAGACCuAuGuUC dTsdT dTsdT HAMP AD-48189 382 A-18280.11 GAAcAuAGGucuuGGAAuA A-100215.1 UAUUCCAAGACcuAuGuUc dTsdT dTsdT HAMP AD-48148 382 A-100179.11 GAAcAuAGGucuuGGAAuA A-100189.1 UAUUCCAAGACCuAuGuUc dTdT dTdT HAMP AD-48215 382 A-18280.8 GAAcAuAGGucuuGGAAuA A-100212.1 UAuUCcAAGACCuAuGuUC dTsdT dTsdT HAMP AD-48218 382 A-100179.26 GAAcAuAGGucuuGGAAuA A-100232.1 uAuUCCAAgAcCuAuGuuc dTdT dTdT HAMP AD-48135 382 A-100179.1 GAAcAuAGGucuuGGAAuA A-100180.1 uAuUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-47004 382 A-98326.1 GAACfAUfAGGUfCfUfUf A-98327.1 CfAUUCCfAAGACCCfAUG GGAAUfAdTsdT UUCdTsdT HAMP AD-48194 382 A-18280.12 GAAcAuAGGucuuGGAAuA A-100216.1 uAUUCCAAGACCuAuGuUC dTsdT dTsdT HAMP AD-48197 382 A-100239.1 GAAcAuAGGuCdTUdGGdA A-100240.1 dTAdTUdCCdAAdGACCuA AdTAdTdT uGuucdTdT HAMP AD-11459 382 A-18280.2 GAAcAuAGGucuuGGAAuA A-18304.1 uAuUCcAAGACCuAuGuUC dTsdT dTsdT HAMP AD-48164 382 A-100179.16 GAAcAuAGGucuuGGAAuA A-100201.1 uAUUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48158 382 A-100179.15 GAAcAuAGGucuuGGAAuA A-100200.1 uAUUCCAAGACCuAuGuUC dTdT dTdT HAMP AD-48204 382 A-100208.1 GAAcAcAGGucuuGGAAuA A-100209.1 uAuUCcAAGACCuGuGuUC dTdT dTdT HAMP AD-48181 382 A-100192.2 GAAcAuAGGucuUGGAAUA A-100180.7 uAuUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48223 382 A-100233.1 GAAcAuAGGucuuGGAAuA A-100234.1 uAuUCcAAGACCuAuGuUC uu uu HAMP AD-48190 382 A-100179.21 GAAcAuAGGucuuGGAAuA A-100227.1 uAuUCCAAGaCCuAuGuuc dTdT dTdT HAMP AD-48163 382 A-100195.5 GAAcAuAGGUCUUGGAAUA A-100180.4 uAuUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48140 382 A-100191.2 GAAcAuAGGucuuGGAAUA A-100180.8 uAuUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48169 382 A-100194.9 GAAcAuAGGuCUUGGAAUA A-100180.5 uAuUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48220 382 A-18280.9 GAAcAuAGGucuuGGAAuA A-100213.1 uAuUCcAAGACCuAuGuUc dTsdT dTsdT HAMP AD-48184 382 A-15167.3 GAACAUAGGUCUUGGAAUA A-18304.6 uAuUCcAAGACCuAuGuUC dTsdT dTsdT HAMP AD-48176 382 A-100179.18 GAAcAuAGGucuuGGAAuA A-100203.1 uAUUCCAAGACCuAuGuUc dTdT dTdT HAMP AD-48175 382 A-100193.2 GAAcAuAGGucUUGGAAUA A-100180.6 uAuUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48146 382 A-100179.13 GAAcAuAGGucuuGGAAuA A-100198.1 uAUUCCAAGACCUAUGUUc dTdT dTdT HAMP AD-48182 382 A-100179.19 GAAcAuAGGucuuGGAAuA A-100204.1 uAuUCcAAGACCuAuGuUc dTdT dTdT HAMP AD-48199 382 A-100207.1 GAAcAuAGGUCUUGGAAuA A-100180.10 uAuUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48157 382 A-100196.3 GAAcAUAGGUCUUGGAAUA A-100180.3 uAuUCcAGACCuAuGuUC dTdT dTdT HAMP AD-48206 382 A-100219.1 GAAcAcAGGucuuGGAAuA A-100220.1 uAuUCcAAGACCuGuGuUC dTsdT dTsdT HAMP AD-48193 382 A-100206.1 GAAcAuAGGuCuuGGAAuA A-100180.9 uAuUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48152 382 A-100179.14 GAAcAuAGGucuuGGAAuA A-100199.1 uAUUCCAAGACCUAUGUUC dTdT dTdT HAMP AD-48151 382 A-98135.7 GAACAUAGGUCUUGGAAUA A-100180.2 uAuUCcAAGACCuAuGuUC dTdT dTdT HAMP AD-48170 382 A-100179.17 GAAcAuAGGucuuGGAAuA A-100202.1 uAuUCUAAGACCuAuGuUC dTdT dTdT HAMP AD-47010 383 A-98328.1 AACfAUfAGGUfCfUfUfG A-98329.1 UCfAUUCCfAAGACCCfAU GAAUfAAdTdT GUUdTdT HAMP AD-45106 383 A-94224.1 AAcAuAGGucuuGGAAuAA A-94225.1 UuAUUCcAAGACCuAUGUU dTsdT dTsdT HAMP AD-48222 385 A-100224.1 AAcAuAGGucuuGGAAuAA A-100225.1 UuAUUCcAAGACCuAuGUU dTsdT dTsdT HAMP AD-48217 385 A-100221.2 cAuAGGucuuGGAAuAAAA A-100223.1 UUUuAUUCcAAGACCuAU dTdT dTdT HAMP AD-48185 385 A-100221.3 cAuAGGucuuGGAAuAAAA A-100226.1 uUUUAuuCCaaGACCUaug dTdT dTdT HAMP AD-48212 385 A-100221.1 cAuAGGucuuGGAAuAAAA A-100222.1 UuUuAuUCcAAGACCuAuG dTdT dTdT HAMP AD-48198 396 A-100252.1 GAAuAAAAuGGcuGGuucu A-100253.1 AGAACcAGCcAUUUuAUUC dTdT dTdT HAMP AD-48209 396 A-100252.3 GAAuAAAAuGGcuGGuucu A-100255.1 AGAACCAGcCaUuUuAuUc dTdT dTdT HAMP AD-48203 396 A-100252.2 GAAuAAAAuGGcuGGuucu A-100254.1 AGAACcAGCCAuuUUAuuc dTdT dTdT HAMP AD-47015 396 A-98330.1 GAAUfAAAAUfGGCfUfGG A-98331.1 AGAACCfAGCCfAUUUCfA UfUfCfUfdTsdT UUCdTsdT HAMP AD-45112 396 A-94226.1 GAAuAAAAuGGcuGGuucu A-94227.1 AGAACcAGCcAUUUuAUUC dTsdT dTsdT HAMP AD-47020 398 A-98332.1 AUfAAAAUfGGCfUfGGUf A-98333.1 AAAGAACCfAGCCfAUUUC UfCfUfUfUfdTsdT fAUdTsdT HAMP AD-45118 398 A-94228.1 AuAAAAuGGcuGGuucuuu A-94229.1 AAAGAACcAGCcAUUUuAU dTsdT dTsdT HAMP AD-47025 399 A-98334.1 UfAAAAUfGGCfUfGGUfU A-98335.1 AAAAGAACCfAGCCfAUUU fCfUfUfUfUfdTsdT CfAdTsdT HAMP AD-45077 399 A-94230.1 uAAAAuGGcuGGuucuuuu A-94231.1 AAAAGAACcAGCcAUUUuA dTsdT dTsdT HAMP AD-47030 402 A-98336.1 AAUfGGCfUfGGUfUfCfU A-98337.1 AACfAAAAGAACCfAGCCf fUfUfUfGUfUfdTsdT AUUdTsdT HAMP AD-45083 402 A-94232.1 AAuGGcuGGuucuuuuGuu A-94233.1 AAcAAAAGAACcAGCcAUU dTsdT dTsdT HAMP AD-46993 403 A-98338.1 AUfGGCfUfGGUfUfCfUf A-98339.1 AAACfAAAAGAACCfAGCC UfUfUfGUfUfUfdTsdT fAUdTsdT HAMP AD-45089 403 A-94234.1 AuGGcuGGuucuuuuGuuu A-94235.1 AAAcAAAAGAACcAGCcAU dTsdT dTsdT HAMP AD-46999 407 A-98340.1 CfUfGGUfUfCfUfUfUfU A-98341.1 UGGAAAACfAAAAGAACCf fGUfUfUfUfCfCfAdTsdT AGdTsdT It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.

Table 5: HAMP Unmodified Sequences

TABLE 5 Start Antisense SEQ ID Sense SEQ ID Target Duplex ID Position Name Antisense Sequence NO Name Sense Sequence NO HAMP AD-47121 62 A-98153.1 AGACGGCACGAUGG A-98154.1 AAGUGCCAUCGU CACUUdTdT GCCGUCUdTdT HAMP AD-47133 67 A-98157.1 GCACGAUGGCACUG A-98158.1 AAGCUCAGUGCCA AGCUUdTdT UCGUGCdTdT HAMP AD-47127 67 A-98155.1 GCACGAUGGCACUG A-98156.1 UAGCUCAGUGCC AGCUAdTdT AUCGUGCdTdT HAMP AD-47145 74 A-98161.1 GGCACUGAGCUCCCA A-98162.1 AAUCUGGGAGCU GAUUdTdT CAGUGCCdTdT HAMP AD-47139 74 A-98159.1 GGCACUGAGCUCCCA A-98160.1 UAUCUGGGAGCU GAUAdTdT CAGUGCCdTdT HAMP AD-47157 76 A-98165.1 CACUGAGCUCCCAGA A-98166.1 AAGAUCUGGGAG UCUUdTdT CUCAGUGdTdT HAMP AD-47151 76 A-98163.1 CACUGAGCUCCCAGA A-98164.1 UAGAUCUGGGAG UCUAdTdT CUCAGUGdTdT HAMP AD-47163 132 A-98167.1 CUGACCAGUGGCUC A-98168.1 AAACAGAGCCACU UGUUUdTdT GGUCAGdTdT HAMP AD-47122 140 A-98169.1 UGGCUCUGUUUUCC A-98170.1 UUGUGGGAAAAC CACAAdTdT AGAGCCAdTdT HAMP AD-47128 146 A-98171.1 UGUUUUCCCACAAC A-98172.1 UGUCUGUUGUGG AGACAdTdT GAAAACAdTdT HAMP AD-47134 146 A-98173.1 UGUUUUCCCACAAC A-98174.1 AGUCUGUUGUGG AGACUdTdT GAAAACAdTdT HAMP AD-47140 155 A-98175.1 ACAACAGACGGGACA A-98176.1 AAGUUGUCCCGU ACUUdTdT CUGUUGUdTdT HAMP AD-47152 157 A-98179.1 AACAGACGGGACAAC A-98180.1 ACAAGUUGUCCC UUGUdTdT GUCUGUUdTdT HAMP AD-47146 157 A-98177.1 AACAGACGGGACAAC A-98178.1 UCAAGUUGUCCC UUGAdTdT GUCUGUUdTdT HAMP AD-47158 160 A-98181.1 AGACGGGACAACUU A-98182.1 UCUGCAAGUUGU GCAGAdTdT CCCGUCUdTdT HAMP AD-47164 161 A-98183.1 GACGGGACAACUUG A-98184.1 UUCUGCAAGUUG CAGAAdTdT UCCCGUCdTdT HAMP AD-47123 161 A-98185.1 GACGGGACAACUUG A-98186.1 AUCUGCAAGUUG CAGAUdTdT UCCCGUCdTdT HAMP AD-47135 162 A-98189.1 ACGGGACAACUUGC A-98190.1 ACUCUGCAAGUU AGAGUdTdT GUCCCGUdTdT HAMP AD-47129 162 A-98187.1 ACGGGACAACUUGC A-98188.1 UCUCUGCAAGUU AGAGAdTdT GUCCCGUdTdT HAMP AD-47141 242 A-98191.1 GAGGCGAGACACCCA A-98192.1 UAAGUGGGUGUC CUUAdTdT UCGCCUCdTdT HAMP AD-47147 242 A-98193.1 GAGGCGAGACACCCA A-98194.1 AAAGUGGGUGUC CUUUdTdT UCGCCUCdTdT HAMP AD-47153 253 A-98195.1 CCCACUUCCCCAUCU A-98196.1 AUGCAGAUGGGG GCAUdTdT AAGUGGGdTdT HAMP AD-47159 258 N-98197.1 UUCCCCAUCUGCAU A-98198.1 AGAAAAUGCAGA UUUCUdTdT UGGGGAAdTdT HAMP AD-47165 261 A-98199.1 CCCAUCUGCAUUUU A-98200.1 AGCAGAAAAUGC CUGCUdTdT AGAUGGGdTdT HAMP AD-47124 275 A-98201.1 CUGCUGCGGCUGCU A-98202.1 AUGACAGCAGCCG GUCAUdTdT CAGCAGdTdT HAMP AD-47136 276 A-98205.1 UGCUGCGGCUGCUG A-98206.1 AAUGACAGCAGCC UCAUUdTdT GCAGCAdTdT HAMP AD-47130 276 A-98203.1 UGCUGCGGCUGCUG A-98204.1 UAUGACAGCAGCC UCAUAdTdT GCAGCAdTdT HAMP AD-47142 278 A-98207.1 CUGCGGCUGCUGUC A-98208.1 UCGAUGACAGCA AUCGAdTdT GCCGCAGdTdT HAMP AD-47148 279 A-98209.1 UGCGGCUGCUGUCA A-98210.1 AUCGAUGACAGC UCGAUdTdT AGCCGCAdTdT HAMP AD-47160 280 A-98213.1 GCGGCUGCUGUCAU A-98214.1 AAUCGAUGACAG CGAUUdTdT CAGCCGCdTdT HAMP AD-47154 280 A-98211.1 GCGGCUGCUGUCAU A-98212.1 UAUCGAUGACAG CGAUAdTdT CAGCCGCdTdT HAMP AD-47166 281 4-98215.1 CGGCUGCUGUCAUC A-98216.1 UGAUCGAUGACA GAUCAdTdT GCAGCCGdTdT HAMP AD-47125 282 A-98217.1 GGCUGCUGUCAUCG A-98218.1 UUGAUCGAUGAC AUCAAdTdT AGCAGCCdTdT HAMP AD-47131 283 A-98219.1 GCUGCUGUCAUCGA A-98220.1 UUUGAUCGAUGA UCAAAdTdT CAGCAGCdTdT HAMP AD-47137 284 A-98221.1 CUGCUGUCAUCGAU A-98222.1 UUUUGAUCGAUG CAAAAdTdT ACAGCAGdTdT HAMP AD-47143 284 A-98223.1 CUGCUGUCAUCGAU A-98224.1 AUUUGAUCGAUG CAAAUdTdT ACAGCAGdTdT HAMP AD-47149 285 A-98225.1 UGCUGUCAUCGAUC A-98226.1 ACUUUGAUCGAU AAAGUdTdT GACAGCAdTdT HAMP AD-47161 286 A-98229.1 GCUGUCAUCGAUCA A-98230.1 AACUUUGAUCGA AAGUUdTdT UGACAGCdTdT HAMP AD-47155 286 4-98227.1 GCUGUCAUCGAUCA A-98228.1 UACUUUGAUCGA AAGUAdTdT UGACAGCdTdT HAMP AD-47167 287 A-98231.1 CUGUCAUCGAUCAA A-98232.1 ACACUUUGAUCG AGUGUdTdT AUGACAGdTdT HAMP AD-47132 288 A-98235.1 UGUCAUCGAUCAAA A-98236.1 AACACUUUGAUC GUGUUdTdT GAUGACAdTdT HAMP AD-47126 288 A-98233.1 UGUCAUCGAUCAAA A-98234.1 UACACUUUGAUC GUGUAdTdT GAUGACAdTdT HAMP AD-47138 290 A-98237.1 UCAUCGAUCAAAGU A-98238.1 UCCACACUUUGA GUGGAdTdT UCGAUGAdTdT HAMP AD-47144 290 A-98239.1 UCAUCGAUCAAAGU A-98240.1 ACCACACUUUGAU GUGGUdTdT CGAUGAdTdT HAMP AD-47095 291 A-98151.1 CAUCGAUCAAAGUG A-98152.1 UCCCACACUUUGA UGGGAdTdT UCGAUGdTdT HAMP AD-47156 295 A-98243.1 GAUCAAAGUGUGGG A-98244.1 AACAUCCCACACU AUGUUdTdT UUGAUCdTdT HAMP AD-47150 295 A-98241.1 GAUCAAAGUGUGGG A-98242.1 UACAUCCCACACU AUGUAdTdT UUGAUCdTdT HAMP AD-47162 299 A-98245.1 AAAGUGUGGGAUGU A-98246.1 ACAGCACAUCCCA GCUGUdTdT CACUUUdTdT HAMP AD-47078 307 A-98067.1 GGAUGUGCUGCAAG A-98068.1 UACGUCUUGCAG ACGUAdTdT CACAUCCdTdT HAMP AD-47084 309 A-98069.1 AUGUGCUGCAAGAC A-98070.1 UCUACGUCUUGC GUAGAdTdT AGCACAUdTdT HAMP AD-47090 310 A-98071.1 UGUGCUGCAAGACG A-98072.1 UUCUACGUCUUG UAGAAdTdT CAGCACAdTdT HAMP AD-47096 313 A-98073.1 GCUGCAAGACGUAG A-98074.1 AGGUUCUACGUC AACCUdTdT UUGCAGCdTdT HAMP AD-47101 314 A-98075.1 CUGCAAGACGUAGA A-98076.1 UAGGUUCUACGU ACCUAdTdT CUUGCAGdTdT HAMP AD-47106 322 A-98077.1 CGUAGAACCUACCU A-98078.1 AGGGCAGGUAGG GCCCUdTdT UUCUACGdTdT HAMP AD-47111 347 A-98079.1 GUCCCCUCCCUUCCU A-98080.1 AAUAAGGAAGGG UAUUdTdT AGGGGACdTdT HAMP AD-47116 348 A-98081.1 UCCCCUCCCUUCCUU A-98082.1 AAAUAAGGAAGG AUUUdTdT GAGGGGAdTdT HAMP AD-47079 349 A-98083.1 CCCCUCCCUUCCUUA A-98084.1 UAAAUAAGGAAG UUUAdTdT GGAGGGGdTdT HAMP AD-47085 350 A-98085.1 CCCUCCCUUCCUUAU A-98086.1 AUAAAUAAGGAA UUAUdTdT GGGAGGGdTdT HAMP AD-47091 351 A-98087.1 CCUCCCUUCCUUAU A-98088.1 AAUAAAUAAGGA UUAUUdTdT AGGGAGGdTdT HAMP AD-47097 352 A-98089.1 CUCCCUUCCUUAUU A-98090.1 UAAUAAAUAAGG UAUUAdTdT AAGGGAGdTdT HAMP AD-47102 352 A-98091.1 CUCCCUUCCUUAUU A-98092.1 AAAUAAAUAAGG UAUUUdTdT AAGGGAGdTdT HAMP AD-47107 354 A-98093.1 CCCUUCCUUAUUUA A-98094.1 AGGAAUAAAUAA UUCCUdTdT GGAAGGGdTdT HAMP AD-47112 355 A-98095.1 CCUUCCUUAUUUAU A-98096.1 UAGGAAUAAAUA UCCUAdTdT AGGAAGGdTdT HAMP AD-47117 355 A-98097.1 CCUUCCUUAUUUAU A-98098.1 AAGGAAUAAAUA UCCUUdTdT AGGAAGGdTdT HAMP AD-47086 356 A-98101.1 CUUCCUUAUUUAU A-98102.1 ACAGGAAUAAAU CCUGUdTdt AAGGAAGdTdT HAMP AD-47080 356 N-98099.1 CUUCCUUAUUUAUU A-98100.1 UCAGGAAUAAAU CCUGAdTdT AAGGAAGdTdT HAMP AD-47092 357 A-98103.1 UUCCUUAUUUAUUC A-98104.1 AGCAGGAAUAAA CUGCUdTdT UAAGGAAdTdT HAMP AD-47103 358 A-98107.1 UCCUUAUUUAUUCC A-98108.1 AAGCAGGAAUAA UGCUUdTdT AUAAGGAdTdT HAMP AD-47098 358 A-98105.1 UCCUUAUUUAUUCC A-98106.1 UAGCAGGAAUAA UGCUAdTdT AUAAGGAdTdT HAMP AD-47113 359 A-98111.1 CCUUAUUUAUUCCU A-98112.1 ACAGCAGGAAUA GCUGUdTdT AAUAAGGdTdT HAMP AD-47108 359 A-98109.1 CCUUAUUUAUUCCU A-98110.1 UCAGCAGGAAUA GCUGAdTdT AAUAAGGdTdT HAMP AD-47118 363 A-98113.1 AUUUAUUCCUGCUG A-98114.1 UGGGGCAGCAGG CCCCAdTdT AAUAAAUdTdT HAMP AD-47081 365 N-98115.1 UUAUUCCUGCUGCC A-98116.1 UCUGGGGCAGCA CCAGAdTdT GGAAUAAdTdT HAMP AD-47087 366 A-98117.1 UAUUCCUGCUGCCC A-98118.1 UUCUGGGGCAGC CAGAAdTdT AGGAAUAdTdT HAMP AD-47093 369 A-98119.1 UCCUGCUGCCCCAGA A-98120.1 AUGUUCUGGGGC ACAUdTdT AGCAGGAdTdT HAMP AD-47099 370 A-98121.1 CCUGCUGCCCCAGAA A-98122.1 UAUGUUCUGGGG CAUAdTdT CAGCAGGdTdT HAMP AD-47104 373 A-98123.1 GCUGCCCCAGAACAU A-98124.1 ACCUAUGUUCUG AGGUdTdT GGGCAGCdTdT HAMP AD-47109 375 A-98125.1 UGCCCCAGAACAUAG A-98126.1 AGACCUAUGUUC GUCUdTdT UGGGGCAdTdT HAMP AD-47114 376 A-93127.1 GCCCCAGAACAUAGG A-98128.1 AAGACCUAUGUU UCUUdTdT CUGGGGCdTdT HAMP AD-47119 379 A-98129.1 CCAGAACAUAGGUC A-98130.1 UCCAAGACCUAUG UUGGAdTdT UUCUGGdTdT HAMP AD-47082 380 A-98131.1 CAGAACAUAGGUCU A-98132.1 UUCCAAGACCUAU UGGAAdTdT GUUCUGdTdT HAMP AD-47088 381 A-98133.1 AGAACAUAGGUCUU A-98134.1 AUUCCAAGACCUA GGAAUdTdT UGUUCUdTdT HAMP AD-47094 382 A-98135.1 GAACAUAGGUCUUG A-98136.1 UAUUCCAAGACCU GAAUAdTdT AUGUUCdTdT HAMP AD-47094 382 N-98135.1 GAACAUAGGUCUUG A-98136.1 UAUUCCAAGACCU GAAUAdTdT AUGUUCdTdT HAMP AD-48210 382 A-100210.1 GAACACAGGUCUUG A-100211.1 UAUUCCAAGACCU GAAUAdTdT GUGUUCdTdT HAMP AD-47100 383 A-98137.1 AACAUAGGUCUUGG A-98138.1 UUAUUCCAAGACC AAUAAdTdT UAUGUUdTdT HAMP AD-47105 396 A-98139.1 GAAUAAAAUGGCUG A-98140.1 AGAACCAGCCAUU GUUCUdTdT UUAUUCdTdT HAMP AD-47110 398 A-98141.3 AUAAAAUGGCUGGU A-98142.1 AAAGAACCAGCCA UCUUUdTdT UUUUAUdTdT HAMP AD-47115 399 A-98143.1 UAAAAUGGCUGGUU A-98144.1 AAAAGAACCAGCC CUUUUdTdT AUUUUAdTdT HAMP AD-47120 402 A-98145.1 AAUGGCUGGUUCUU A-98146.1 AACAAAAGAACCA UUGUUdTdT GCCAUUdTdT HAMP AD-47083 403 A-98147.1 AUGGCUGGUUCUUU A-98148.1 AAACAAAAGAACC UGUUUdTdT AGCCAUdTdT HAMP AD-47089 407 A-98149.1 CUGGUUCUUUUGUU A-98150.1 UGGAAAACAAAA UUCCAdTdT GAACCAGdTdT

Table 6: HAMP Single Dose Screen (Modified Duplexes, Dual Luciferase Assay)

TABLE 6 Start Posi- 10 nM 0.1 nM 0.01 nM Target Duplex ID tion Avg SD Avg SD Avg SD HAMP AD-45073 2 107.73 0.50 92.94 7.41 HAMP AD-45079 7 110.26 7.14 101.78 5.79 HAMP AD-45085 16 90.81 0.48 96.06 2.19 HAMP AD-29928 43 102.01 15.80 96.01 1.70 HAMP AD-45674 43 94.81 4.68 108.44 7.69 HAMP AD-45680 43 109.80 2.04 111.06 5.64 HAMP AD-45686 48 89.78 15.04 110.29 0.29 HAMP AD-45698 48 103.33 8.83 112.53 1.57 HAMP AD-45692 48 110.03 6.99 115.05 0.14 HAMP AD-45354 51 111.45 7.56 105.64 4.49 HAMP AD-29929 54 99.33 11.26 104.08 6.92 HAMP AD-45091 55 116.71 3.20 102.27 0.81 HAMP AD-29930 59 88.47 0.38 102.18 7.79 HAMP AD-29931 60 104.54 3.36 104.80 3.55 HAMP AD-45704 60 142.74 0.80 122.02 1.37 HAMP AD-45710 60 135.87 3.55 129.05 1.72 HAMP AD-29932 61 103.48 6.29 108.36 1.31 HAMP AD-29933 62 110.13 1.03 104.36 6.68 HAMP AD-45675 62 113.15 1.01 107.56 0.54 HAMP AD-45716 62 111.06 12.39 113.09 8.16 HAMP AD-29934 63 101.68 3.60 96.37 6.01 HAMP AD-29935 64 100.63 8.13 93.98 8.75 HAMP AD-45687 64 103.09 8.83 105.61 3.09 HAMP AD-45681 64 117.87 2.59 111.72 1.69 HAMP AD-29936 66 98.38 12.53 98.56 13.20 HAMP AD-29937 67 93.41 2.34 97.50 10.28 HAMP AD-45699 67 47.01 9.59 98.55 3.80 HAMP AD-45693 67 84.68 3.15 113.79 5.11 HAMP AD-45711 68 113.03 9.72 108.10 3.83 HAMP AD-45717 68 99.40 12.84 110.38 0.04 HAMP AD-45705 68 110.22 3.84 117.90 9.96 HAMP AD-45682 69 96.60 3.60 103.41 1.06 HAMP AD-45688 69 100.44 9.14 104.93 5.18 HAMP AD-45676 69 106.83 9.15 106.73 1.89 HAMP AD-45360 70 92.88 0.12 93.73 2.85 HAMP AD-45366 71 92.46 2.58 99.04 0.39 HAMP AD-29938 72 62.08 21.83 75.55 6.85 HAMP AD-45372 73 59.85 2.76 96.31 5.86 HAMP AD-45700 74 12.85 5.11 63.97 14.79 HAMP AD-29939 74 36.40 19.57 67.18 9.10 HAMP AD-45694 74 17.85 4.97 90.43 0.13 HAMP AD-29940 75 49.83 6.31 76.05 7.08 HAMP AD-45712 76 32.07 2.85 63.27 3.48 HAMP AD-29941 76 81.10 0.03 97.49 9.32 HAMP AD-45706 76 43.48 6.67 97.60 1.61 HAMP AD-45097 88 50.62 0.50 71.18 1.94 HAMP AD-45103 91 53.20 9.02 96.52 7.45 HAMP AD-45378 116 95.96 1.21 103.17 3.99 HAMP AD-45383 117 99.99 2.44 104.79 5.38 HAMP AD-45388 118 98.52 4.10 105.96 3.21 HAMP AD-45393 120 103.62 5.17 102.44 6.38 HAMP AD-45355 121 73.28 0.51 96.56 1.06 HAMP AD-45361 122 98.67 0.23 99.82 4.47 HAMP AD-45367 123 90.48 1.28 102.75 4.07 HAMP AD-45373 126 106.01 8.36 99.38 4.05 HAMP AD-45109 132 85.86 5.55 95.06 3.75 HAMP AD-45115 140 100.97 1.25 90.90 9.28 HAMP AD-45074 142 95.53 2.12 95.37 2.74 HAMP AD-45677 146 58.20 5.06 80.37 5.46 HAMP AD-45683 146 67.80 1.12 88.08 7.96 HAMP AD-45718 146 76.16 3.48 100.37 3.35 HAMP AD-45080 149 52.89 1.28 84.16 0.02 HAMP AD-45379 150 48.97 3.64 103.43 1.19 HAMP AD-29942 151 88.17 4.85 97.49 7.95 HAMP AD-29943 152 43.37 9.93 73.15 23.90 HAMP AD-29944 153 80.38 9.90 92.54 7.85 HAMP AD-45695 153 65.57 2.52 92.72 5.87 HAMP AD-45689 153 72.67 0.78 93.00 2.67 HAMP AD-29945 154 69.81 13.13 76.49 17.11 HAMP AD-29946 155 75.80 1.18 80.39 14.15 HAMP AD-45713 157 70.69 1.76 94.45 0.39 HAMP AD-45707 157 71.62 6.17 94.94 2.22 HAMP AD-45701 157 79.39 1.97 101.46 0.70 HAMP AD-45384 159 89.86 1.67 102.53 2.37 HAMP AD-45389 160 41.14 0.44 90.29 3.94 HAMP AD-45678 161 55.04 0.96 76.03 3.63 HAMP AD-45719 161 55.02 5.94 84.45 2.01 HAMP AD-29947 161 81.45 6.55 89.78 7.92 HAMP AD-45690 162 105.99 3.29 97.21 2.61 HAMP AD-45696 162 105.48 0.08 99.13 0.28 HAMP AD-45684 162 96.14 6.48 104.09 2.52 HAMP AD-30016 163 57.89 8.28 90.60 14.06 HAMP AD-45394 164 87.68 5.01 108.27 2.32 HAMP AD-45702 165 70.60 2.02 93.12 5.12 HAMP AD-45708 165 74.75 3.73 98.51 2.26 HAMP AD-45714 165 73.26 2.24 102.34 12.47 HAMP AD-29949 166 102.90 8.09 91.62 0.16 HAMP AD-45086 167 120.81 3.27 106.79 7.19 HAMP AD-45356 168 81.17 4.40 93.13 0.76 HAMP AD-45685 169 114.45 9.16 98.53 0.41 HAMP AD-45679 169 105.22 9.07 101.56 5.80 HAMP AD-45720 169 121.03 5.25 110.57 1.75 HAMP AD-45703 170 44.33 1.60 79.12 4.45 HAMP AD-45697 170 46.91 2.65 87.12 1.48 HAMP AD-45691 170 54.15 1.94 92.73 6.86 HAMP AD-45362 189 40.88 0.51 88.62 2.54 HAMP AD-45368 190 31.23 1.19 95.59 2.85 HAMP AD-45374 199 101.82 3.03 101.10 0.65 HAMP AD-45092 222 87.17 5.48 98.58 1.36 HAMP AD-45721 228 46.67 6.31 81.09 9.13 HAMP AD-45715 228 49.86 3.40 88.14 4.98 HAMP AD-45709 228 77.17 5.09 98.27 3.31 HAMP AD-45380 230 62.83 3.09 103.76 1.31 HAMP AD-45385 231 98.28 0.86 102.61 0.12 HAMP AD-29950 232 55.13 8.89 67.22 10.51 HAMP AD-45390 233 43.22 3.42 94.61 0.86 HAMP AD-29951 234 37.28 9.48 53.43 13.93 HAMP AD-45395 235 60.56 0.93 96.88 1.63 HAMP AD-45727 239 41.79 5.36 73.07 5.68 HAMP AD-45732 239 40.15 8.90 73.60 14.88 HAMP AD-29952 239 97.66 18.17 104.87 4.70 HAMP AD-29953 240 86.68 10.48 88.35 7.38 HAMP AD-30017 241 33.76 16.25 60.73 30.76 HAMP AD-30018 242 41.44 14.83 70.97 23.75 HAMP AD-45737 242 17.97 4.49 71.13 9.76 HAMP AD-29956 246 89.56 4.13 97.82 5.97 HAMP AD-45357 247 82.69 2.17 93.22 4.53 HAMP AD-45363 248 93.32 5.91 91.24 0.20 HAMP AD-45747 251 70.65 8.85 97.73 2.26 HAMP AD-45752 251 89.51 3.39 98.35 2.19 HAMP AD-45757 251 82.94 6.75 102.34 3.76 HAMP AD-29957 252 81.99 11.58 93.00 9.35 HAMP AD-45399 253 64.38 0.64 97.91 2.54 HAMP AD-45098 255 82.48 2.01 76.84 2.07 HAMP AD-45400 256 41.85 0.69 73.87 3.09 HAMP AD-45381 257 33.48 1.75 76.90 0.24 HAMP AD-45401 258 20.19 1.67 47.65 3.20 HAMP AD-29958 261 56.65 14.92 84.66 28.04 HAMP AD-45391 262 24.94 0.82 89.45 2.12 HAMP AD-29959 267 31.48 11.63 63.85 30.99 HAMP AD-29960 268 79.91 12.47 93.49 6.68 HAMP AD-30019 270 63.27 10.61 74.99 18.30 HAMP AD-45396 271 119.24 2.30 111.41 3.10 HAMP AD-45358 272 94.71 8.18 101.07 2.21 HAMP AD-45364 273 84.76 0.19 96.53 5.62 HAMP AD-29962 274 76.15 9.81 86.71 13.14 HAMP AD-45370 275 72.03 3.07 96.93 2.90 HAMP AD-45728 276 16.69 2.26 33.05 13.53 HAMP AD-45722 276 14.19 2.15 36.38 8.98 HAMP AD-29963 276 33.66 10.79 68.80 33.38 HAMP AD-45104 278 68.72 2.74 87.22 0.49 HAMP AD-29964 279 71.02 18.87 76.03 27.29 HAMP AD-45738 280 50.02 8.64 70.44 7.26 HAMP AD-45733 280 57.29 5.28 84.47 5.76 HAMP AD-29965 281 55.85 8.35 72.34 23.30 HAMP AD-30020 283 68.86 8.88 66.02 23.24 HAMP AD-45748 284 21.85 1.77 35.95 6.98 HAMP AD-45743 284 29.01 1.73 42.99 5.69 HAMP AD-30021 284 42.30 7.75 66.28 27.47 HAMP AD-11441 285 15.04 8.59 34.60 10.87 63.42 16.67 HAMP AD-45758 286 17.08 0.43 33.34 3.43 HAMP AD-45753 286 25.19 4.02 80.83 6.73 HAMP AD-29968 286 57.05 12.26 85.22 13.56 HAMP AD-29969 287 81.97 16.19 102.53 21.58 HAMP AD-45729 288 9.67 1.06 32.83 13.93 HAMP AD-45723 288 20.87 3.89 66.57 4.73 HAMP AD-29970 288 65.21 1.72 84.12 5.99 HAMP AD-45744 290 40.34 1.92 56.56 3.09 HAMP AD-45739 290 29.46 2.77 67.24 9.80 HAMP AD-45734 290 53.39 2.32 83.49 1.19 HAMP AD-11436 291 19.18 8.22 42.74 14.18 76.43 23.00 HAMP AD-29971 291 29.02 9.90 52.08 14.09 HAMP AD-45376 292 47.54 0.51 87.50 1.26 HAMP AD-45382 293 37.05 0.44 93.25 4.03 HAMP AD-29972 294 32.08 7.08 53.51 17.12 HAMP AD-45754 295 30.78 4.04 62.74 0.50 HAMP AD-45749 295 48.49 11.81 92.97 1.08 HAMP AD-29973 295 101.69 8.46 97.65 8.24 HAMP AD-45730 296 78.23 2.00 86.13 5.69 HAMP AD-45724 296 82.07 4.46 86.67 2.23 HAMP AD-45759 296 92.10 6.68 97.71 2.40 HAMP AD-45110 297 69.77 2.74 90.01 2.11 HAMP AD-45387 298 98.34 5.75 108.20 1.85 HAMP AD-45740 299 124.12 4.66 101.03 6.78 HAMP AD-45745 299 131.69 10.22 103.23 1.76 HAMP AD-45735 299 111.96 3.19 103.86 6.46 HAMP AD-29974 300 34.04 6.78 53.98 24.23 HAMP AD-29975 301 53.80 12.05 67.73 22.08 HAMP AD-45116 306 25.93 2.21 55.49 5.82 HAMP AD-45075 307 19.84 2.98 63.83 1.45 HAMP AD-45081 309 14.66 0.55 38.67 3.25 HAMP AD-45087 310 11.95 0.17 29.23 1.61 HAMP AD-45093 313 14.07 0.68 45.44 2.30 HAMP AD-45099 322 108.37 1.23 107.51 2.82 HAMP AD-45105 369 102.93 6.40 101.31 4.27 HAMP AD-45111 370 117.00 3.72 104.04 2.33 HAMP AD-45117 373 99.33 0.61 102.95 0.52 HAMP AD-45076 375 62.84 4.39 90.32 1.71 HAMP AD-45082 376 75.58 1.59 95.17 0.21 HAMP AD-45088 379 83.27 12.84 101.84 1.83 HAMP AD-45094 380 99.51 2.23 102.51 2.51 HAMP AD-45100 381 112.68 6.11 107.46 7.75 HAMP AD-45106 383 138.19 1.98 112.49 0.89 HAMP AD-45112 396 128.11 6.65 106.21 3.64 HAMP AD-45118 398 116.86 12.51 103.78 4.53 HAMP AD-45077 399 98.64 1.22 103.30 2.55 HAMP AD-45083 402 114.82 2.26 104.50 0.17 HAMP AD-45089 403 107.59 7.70 103.43 1.69 Data are expressed as percent of mock or AD-1955.

Table 7: HAMP Single Dose Screen (Unmodified Duplexes, Human Endogenous)

TABLE 7 Start Posi- 10 nM 0.1 nM 0.01 nM Target Duplex ID tion Avg SD Avg SD Avg SD HAMP AD-47121 62 22.18 1.49 60.31 16.26 HAMP AD-47133 67 27.53 1.12 53.58 2.92 HAMP AD-47127 67 20.45 4.63 54.16 7.57 HAMP AD-47145 74 19.51 6.65 54.67 10.88 HAMP AD-47139 74 20.72 0.17 58.12 2.90 HAMP AD-47157 76 10.07 0.35 28.24 6.49 HAMP AD-47151 76 12.08 1.83 33.95 2.14 HAMP AD-47163 132 8.58 0.51 44.65 17.97 HAMP AD-47122 140 25.66 0.45 72.63 2.29 HAMP AD-47128 146 30.88 3.04 64.36 2.04 HAMP AD-47134 146 48.07 0.91 72.33 12.48 HAMP AD-47140 155 15.20 1.25 34.69 0.30 HAMP AD-47152 157 13.21 6.55 28.17 1.17 HAMP AD-47146 157 14.77 0.68 32.02 1.58 HAMP AD-47158 160 9.73 1.66 32.92 1.87 HAMP AD-47164 161 5.71 0.44 32.90 2.89 HAMP AD-47123 161 7.88 3.02 39.31 19.09 HAMP AD-47135 162 26.84 0.87 74.06 15.78 HAMP AD-47129 162 27.18 1.57 83.62 13.59 HAMP AD-47141 242 110.80 16.98 127.17 42.39 HAMP AD-47147 242 116.01 9.55 132.52 28.61 HAMP AD-47153 253 34.69 7.47 66.88 2.08 HAMP AD-47159 258 33.41 1.23 57.26 7.97 HAMP AD-47165 261 25.12 0.71 85.70 6.86 HAMP AD-47124 275 36.35 7.66 87.65 11.44 HAMP AD-47136 276 6.06 0.70 40.72 13.94 HAMP AD-47130 276 8.76 0.58 46.31 13.29 HAMP AD-47142 278 24.10 2.89 56.75 18.44 HAMP AD-47148 279 19.36 1.09 57.95 18.21 HAMP AD-47160 280 5.75 0.73 35.24 4.98 HAMP AD-47154 280 15.01 3.91 36.32 0.45 HAMP AD-47166 281 11.98 0.47 51.40 12.88 HAMP AD-47125 282 14.62 1.15 54.37 11.47 HAMP AD-47131 283 8.74 0.45 42.66 12.21 HAMP AD-47137 284 9.97 0.73 36.35 5.96 HAMP AD-47143 284 9.66 0.84 39.72 8.37 HAMP AD-47149 285 13.85 0.47 47.21 9.13 HAMP AD-47161 286 7.28 0.89 31.75 8.03 HAMP AD-47155 286 8.27 0.74 36.03 14.37 HAMP AD-47167 287 8.98 0.14 41.61 6.60 HAMP AD-47132 288 9.08 0.17 38.01 4.01 HAMP AD-47126 288 8.59 3.66 40.28 10.49 HAMP AD-47138 290 41.75 1.27 81.70 14.64 HAMP AD-47144 290 60.81 13.34 107.58 15.10 HAMP AD-47095 291 34.79 5.48 58.98 7.36 HAMP AD-47156 295 39.09 4.26 90.08 4.38 HAMP AD-47150 295 53.01 9.58 99.42 9.61 HAMP AD-47162 299 122.90 8.44 123.74 15.71 HAMP AD-47078 307 26.81 9.00 59.79 15.73 HAMP AD-47084 309 31.16 5.91 59.33 19.95 HAMP AD-47090 310 15.07 5.19 49.74 11.70 HAMP AD-47096 313 49.34 9.86 68.64 0.71 HAMP AD-47101 314 13.36 5.68 38.13 10.64 HAMP AD-47106 322 29.91 1.99 61.30 1.25 HAMP AD-47111 347 21.57 4.45 46.65 10.06 HAMP AD-47116 348 32.95 7.34 65.00 10.06 HAMP AD-47079 349 10.10 2.16 24.36 4.46 HAMP AD-47085 350 8.08 5.13 20.39 9.47 HAMP AD-47091 351 20.73 6.86 42.28 5.15 HAMP AD-47097 352 10.57 2.97 24.58 3.18 HAMP AD-47102 352 15.48 7.81 25.60 8.37 HAMP AD-47107 354 50.89 12.39 61.80 6.52 HAMP AD-47112 355 42.93 6.42 53.00 3.93 HAMP AD-47117 355 33.82 2.18 60.78 7.57 HAMP AD-47086 356 16.50 3.69 34.88 9.79 HAMP AD-47080 356 13.76 3.39 38.95 7.09 HAMP AD-47092 357 35.01 5.39 48.61 6.81 HAMP AD-47103 356 45.09 9.10 66.18 7.81 HAMP AD-47098 358 63.20 11.74 70.69 1.23 HAMP AD-47113 359 27.42 9.95 49.88 7.22 HAMP AD-47108 359 30.30 9.89 52.33 12.30 HAMP AD-47118 363 7.45 0.35 19.20 1.31 HAMP AD-47081 365 4.25 1.97 22.94 6.70 HAMP AD-47087 366 9.49 2.18 37.51 12.04 HAMP AD-47093 369 4.75 1.38 23.36 3.30 HAMP AD-47099 370 5.05 0.01 17.71 7.66 HAMP AD-47104 373 32.32 8.82 37.72 8.15 HAMP AD-47109 375 25.45 4.46 35.56 1.25 HAMP AD-47114 376 10.65 4.55 17.30 6.01 HAMP AD-47119 379 7.99 0.50 17.44 6.45 HAMP AD-47082 380 13.13 1.08 27.19 8.88 HAMP AD-47088 381 5.80 2.75 12.26 7.13 HAMP AD-47094 382 5.59 2.35 11.95 7.39 HAMP AD-47094 382 8.63 3.05 14.02 2.30 22.83 0.56 HAMP AD-48210 382 7.43 7.88 17.06 4.17 30.21 0.63 HAMP AD-47100 383 3.80 2.75 8.41 3.86 HAMP AD-47105 396 6.56 2.25 12.11 4.90 HAMP AD-47110 398 10.42 5.14 21.44 0.24 HAMP AD-47115 399 4.86 0.27 9.25 1.57 HAMP AD-47120 402 5.78 0.12 15.68 0.67 HAMP AD-47083 403 4.36 1.88 14.49 5.26 HAMP AD-47089 407 17.68 1.22 21.61 6.91 Data are expressed as percent of mock.

Table 8: HAMP Single Dose Screen (Modified Duplexes, Human Endogenous)

TABLE 8 Start Posi- 10 nM 0.1 nM 0.01 nM Target Duplex ID tion Avg SD Avg SD Avg SD HAMP AD-47031 62 15.53 6.23 42.68 4.24 HAMP AD-47043 67 21.87 1.62 50.05 4.67 HAMP AD-47037 67 23.85 4.92 53.84 0.43 HAMP AD-47055 74 31.38 2.06 59.08 4.48 HAMP AD-47049 74 30.11 3.10 64.35 3.66 HAMP AD-47067 76 8.71 1.38 28.60 2.87 HAMP AD-47061 76 11.78 3.07 29.53 0.18 HAMP AD-47032 140 37.89 8.04 60.90 3.74 HAMP AD-47038 146 33.92 2.79 53.24 7.14 HAMP AD-47044 146 39.99 7.45 60.74 5.99 HAMP AD-47050 155 14.55 1.46 35.51 0.33 HAMP AD-47062 157 13.42 1.10 45.34 3.82 HAMP AD-47056 157 23.31 0.44 46.02 0.80 HAMP AD-47068 160 24.68 0.67 56.12 4.66 HAMP AD-47033 161 11.56 4.54 36.94 0.19 HAMP AD-47074 161 9.99 1.07 44.47 0.95 HAMP AD-47039 162 63.29 2.38 80.39 15.70 HAMP AD-47045 162 86.89 5.22 96.60 14.33 HAMP AD-47057 242 66.74 2.06 82.90 10.89 HAMP AD-47051 242 72.68 0.12 86.34 3.07 HAMP AD-47063 253 26.21 0.40 58.94 7.25 HAMP AD-47069 258 30.01 3.26 41.02 3.03 HAMP AD-47075 261 30.80 2.74 75.66 1.48 HAMP AD-47034 275 54.15 10.01 75.48 16.26 HAMP AD-47046 276 13.55 0.80 30.18 6.37 HAMP AD-47040 276 18.09 3.87 40.15 14.54 HAMP AD-47058 279 36.00 4.98 64.23 1.93 HAMP AD-47070 280 12.74 1.13 34.84 9.02 HAMP AD-47064 280 17.08 0.13 49.50 0.21 HAMP AD-47076 281 12.07 1.81 36.35 5.58 HAMP AD-47035 282 31.01 7.32 61.60 1.15 HAMP AD-47041 283 16.92 0.64 39.03 10.75 HAMP AD-47053 284 10.31 0.77 23.40 7.24 HAMP AD-47047 284 12.12 0.18 30.96 7.74 HAMP AD-47059 285 20.79 0.79 45.23 6.52 HAMP AD-47071 286 15.36 1.48 36.67 8.67 HAMP AD-47065 286 19.45 0.16 53.77 19.91 HAMP AD-47077 287 9.85 0.40 45.43 2.39 HAMP AD-48208 288 9.71 4.88 14.16 3.25 40.26 4.14 HAMP AD-47042 288 9.47 2.61 24.02 11.39 HAMP AD-48202 288 11.49 3.71 27.05 3.20 69.29 1.70 HAMP AD-47036 288 10.22 1.87 38.40 8.79 HAMP AD-47048 290 38.00 2.44 80.14 9.40 HAMP AD-47054 290 46.82 5.24 87.19 6.81 HAMP AD-47005 291 34.54 2.08 63.87 11.34 HAMP AD-11436 291 43.37 7.53 74.23 14.15 HAMP AD-47066 295 37.84 6.67 66.36 3.03 HAMP AD-47060 295 52.68 4.93 83.68 16.47 HAMP AD-47072 299 74.58 22.86 117.51 7.68 HAMP AD-46988 307 39.46 7.63 78.38 1.82 HAMP AD-46994 309 91.00 6.12 100.96 9.30 HAMP AD-47000 310 42.88 7.35 65.34 6.09 HAMP AD-47006 313 27.81 0.36 71.03 9.01 HAMP AD-47011 314 24.50 4.38 63.98 14.14 HAMP AD-47016 322 65.73 3.26 90.84 9.34 HAMP AD-47021 347 80.76 0.51 86.40 9.24 HAMP AD-47026 348 71.64 5.09 81.58 5.61 HAMP AD-46989 349 90.45 10.46 99.05 11.53 HAMP AD-46995 350 20.68 3.39 75.89 9.25 HAMP AD-47001 351 74.47 1.50 80.49 21.50 HAMP AD-47012 352 71.82 13.01 84.03 5.63 HAMP AD-47007 352 82.28 15.46 89.63 9.85 HAMP AD-47017 354 66.26 8.83 100.80 21.89 HAMP AD-47022 355 63.73 4.49 87.39 9.12 HAMP AD-47027 355 68.87 6.64 108.08 32.59 HAMP AD-46996 355 37.91 1.83 48.04 6.32 HAMP AD-46990 356 41.87 4.92 54.43 6.70 HAMP AD-47002 357 16.19 0.33 42.98 3.19 HAMP AD-47013 358 22.95 0.97 44.27 7.76 HAMP AD-47008 358 20.38 2.71 50.46 16.50 HAMP AD-47023 359 77.40 10.19 95.51 9.29 HAMP AD-47018 359 95.24 14.37 97.19 8.72 HAMP AD-47028 363 28.25 2.86 62.93 4.48 HAMP AD-46991 365 15.53 2.49 29.41 0.30 HAMP AD-46997 366 31.51 3.85 48.07 6.21 HAMP AD-47003 369 9.85 2.64 34.31 6.01 HAMP AD-47009 370 6.69 1.11 24.11 3.57 HAMP AD-47014 373 55.85 3.19 60.89 10.51 HAMP AD-47019 375 28.54 1.87 49.45 14.83 HAMP AD-48214 376 12.63 3.25 16.69 1.38 29.21 0.40 HAMP AD-48219 376 15.92 0.02 18.92 0.48 33.17 2.16 HAMP AD-47024 376 19.61 1.81 42.20 5.93 HAMP AD-48224 379 22.20 5.60 33.45 1.62 52.72 1.81 HAMP AD-48187 379 25.57 5.25 46.92 0.04 73.94 0.20 HAMP AD-47029 379 24.31 0.26 47.37 6.54 HAMP AD-48192 379 19.69 0.78 55.32 4.62 88.04 0.76 HAMP AD-46992 380 23.41 3.32 37.52 4.13 HAMP AD-46998 381 26.55 2.19 49.95 0.87 HAMP AD-48137 382 8.66 0.33 11.24 1.02 26.89 1.08 HAMP AD-48196 382 6.92 3.59 11.81 1.33 22.33 0.98 HAMP AD-48195 382 6.10 2.66 12.50 3.26 26.60 1.17 HAMP AD-48201 382 12.95 1.61 13.01 2.07 25.66 6.95 HAMP AD-48207 382 7.91 2.77 13.17 0.62 21.22 1.42 HAMP AD-48159 382 15.26 0.13 13.59 1.45 28.89 4.67 HAMP AD-48147 382 14.28 0.17 13.65 0.38 25.68 3.81 HAMP AD-48161 382 9.61 3.69 13.77 1.67 22.95 0.59 HAMP AD-48172 382 11.67 0.41 13.98 1.39 27.28 5.22 HAMP AD-48156 382 12.14 0.96 14.06 1.75 28.85 2.18 HAMP AD-48195 382 6.81 0.62 14.14 0.37 27.99 0.61 HAMP AD-48136 382 10.81 4.42 14.16 1.41 29.89 1.13 HAMP AD-48166 382 8.74 2.41 14.22 0.42 25.21 2.09 HAMP AD-48213 382 8.35 1.61 14.49 3.71 22.38 0.33 HAMP AD-48173 382 12.84 3.32 14.51 0.96 27.87 5.30 HAMP AD-48154 382 9.93 2.07 14.80 0.75 23.47 0.94 HAMP AD-48141 382 12.73 2.32 14.92 0.32 26.97 5.76 HAMP AD-48216 382 10.39 0.95 15.18 2.11 22.38 1.02 HAMP AD-48180 382 7.71 0.47 15.20 1.30 29.01 1.11 HAMP AD-48143 382 7.00 2.73 15.44 1.88 24.35 2.09 HAMP AD-48142 382 10.10 3.42 15.50 0.90 25.84 2.08 HAMP AD-48221 382 9.54 1.43 15.56 0.24 23.59 1.06 HAMP AD-48171 382 13.46 5.06 15.67 0.63 26.44 6.58 HAMP AD-48145 382 10.87 0.68 15.70 2.69 28.65 2.93 HAMP AD-48160 382 10.77 0.26 15.74 1.29 28.12 4.11 HAMP AD-48144 382 9.88 0.46 15.75 0.94 33.60 0.84 HAMP AD-48167 382 10.83 1.48 15.87 3.03 24.90 2.15 HAMP AD-48177 382 15.05 2.15 15.87 0.86 28.68 8.05 HAMP AD-48153 382 12.07 5.83 15.92 1.04 31.19 3.85 HAMP AD-48178 382 11.02 0.05 16.06 0.45 27.12 2.27 HAMP AD-48155 382 12.92 0.25 16.32 4.70 27.64 0.32 HAMP AD-48174 382 11.50 2.09 16.39 0.74 27.90 4.46 HAMP AD-48205 382 7.46 7.62 16.39 2.29 24.17 1.56 HAMP AD-48179 382 10.80 3.42 16.50 0.71 27.82 3.57 HAMP AD-48168 382 12.14 4.14 16.63 1.58 27.25 1.24 HAMP AD-48149 382 10.42 0.41 16.71 3.88 28.30 1.91 HAMP AD-48211 382 9.46 4.30 16.80 1.44 25.08 0.20 HAMP AD-48200 382 9.05 1.30 16.97 2.20 28.99 0.38 HAMP AD-48188 382 11.09 3.14 16.99 2.41 32.42 1.58 HAMP AD-48183 382 9.79 11.87 17.20 0.54 42.02 0.63 HAMP AD-48150 382 9.99 8.19 17.30 2.27 35.68 0.32 HAMP AD-48162 382 8.48 2.96 17.38 1.26 29.63 0.85 HAMP AD-48139 382 10.35 1.92 17.78 0.51 36.00 2.79 HAMP AD-9942 382 8.96 1.21 18.03 1.85 30.09 0.13 HAMP AD-48138 382 10.06 2.32 18.04 1.88 26.97 1.56 HAMP AD-11459 382 7.07 0.09 18.93 3.13 HAMP AD-48189 382 8.39 0.33 19.16 0.02 26.30 0.33 HAMP AD-48148 382 10.68 1.22 19.23 3.07 32.33 1.11 HAMP AD-48215 382 12.87 3.87 19.50 0.69 35.59 3.70 HAMP AD-48218 382 12.77 1.57 22.32 2.42 48.01 5.44 HAMP AD-48135 382 13.43 1.45 26.06 4.80 57.92 1.12 HAMP AD-47004 382 10.04 1.61 26.68 3.90 HAMP AD-48194 382 13.40 1.06 27.15 1.41 44.44 2.80 HAMP AD-48197 382 13.40 6.22 27.28 1.85 47.77 5.73 HAMP AD-11459 382 14.32 0.61 28.20 2.18 50.13 0.65 HAMP AD-48164 382 16.52 2.52 28.92 3.09 55.94 0.41 HAMP AD-48158 382 11.63 2.57 29.78 1.47 51.55 0.47 HAMP AD-48204 382 11.53 13.16 29.90 1.81 68.66 2.16 HAMP AD-48181 382 11.31 3.03 30.04 2.07 65.39 3.38 HAMP AD-48223 382 12.35 5.46 30.39 3.74 60.92 1.91 HAMP AD-48190 382 10.09 1.16 30.78 0.29 61.44 0.86 HAMP AD-48163 382 13.43 2.71 31.64 4.03 60.73 1.22 HAMP AD-48140 382 11.77 2.88 31.73 2.21 57.59 0.57 HAMP AD-48169 382 12.50 0.13 32.00 4.43 60.20 3.22 HAMP AD-48220 382 15.52 3.73 32.05 1.48 58.71 2.57 HAMP AD-48184 382 13.23 0.16 33.25 3.63 56.78 2.43 HAMP AD-48176 382 16.68 3.88 34.04 1.12 68.51 2.16 HAMP AD-48175 382 13.60 9.49 34.34 2.07 63.89 2.17 HAMP AD-48146 382 13.16 4.10 35.07 0.23 61.14 1.09 HAMP AD-48182 382 13.71 8.96 36.24 0.98 71.73 0.19 HAMP AD-48199 382 11.16 0.69 36.33 1.26 66.19 0.94 HAMP AD-48157 382 11.27 0.12 36.54 2.61 71.05 0.88 HAMP AD-48206 382 10.51 2.71 36.79 4.47 61.74 0.21 HAMP AD-48193 382 13.00 3.25 37.58 2.12 73.07 0.26 HAMP AD-48152 382 21.49 5.83 39.17 6.15 68.81 10.01 HAMP AD-48151 382 13.62 10.83 39.31 6.55 66.68 1.47 HAMP AD-48170 382 14.54 2.27 47.27 4.01 70.43 2.22 HAMP AD-47010 383 41.47 1.29 64.00 4.87 HAMP AD-48222 385 9.49 0.13 10.82 1.20 18.78 2.86 HAMP AD-48217 385 14.39 3.84 14.26 0.55 19.91 0.71 HAMP AD-48185 385 14.22 4.44 18.35 1.29 37.50 0.03 HAMP AD-48212 385 20.56 4.86 22.61 3.43 26.13 0.41 HAMP AD-48198 396 8.31 23.06 13.10 3.95 50.72 1.39 HAMP AD-48209 396 8.76 5.65 14.85 3.44 33.81 2.06 HAMP AD-48203 396 9.38 2.78 15.08 2.34 35.67 2.38 HAMP AD-47015 396 16.43 2.26 30.67 1.14 HAMP AD-47020 398 50.18 1.59 68.91 17.54 HAMP AD-47025 399 13.04 0.87 19.74 1.31 HAMP AD-47030 402 5.12 0.55 12.72 0.67 HAMP AD-46993 403 5.82 2.21 12.55 1.15 HAMP AD-46999 407 11.34 1.35 15.21 1.41 Data are expressed as percent of mock.

Table 9: HAMP Dose-Response (Dual Luciferase HepG2, Cyno Primary Hepatocytes; Unmodified & Modified Duplexes)

TABLE 9 Start Modification IC50 (nM) Target Duplex ID position status Luc HepG2 Cyno HAMP AD-29939 74 Modified 0.288 HAMP AD-45700 74 Modified 0.752 HAMP AD-29940 75 Modified 0.929 HAMP AD-29943 152 Modified 0.567 HAMP AD-29950 232 Modified 1.527 HAMP AD-29951 234 Modified 0.408 HAMP AD-30017 241 Modified 0.163 HAMP AD-30018 242 Modified 0.517 HAMP AD-29959 267 Modified 0.147 HAMP AD-29963 276 Modified 0.155 HAMP AD-45722 276 Modified 0.299 HAMP AD-29965 281 Modified 1.149 HAMP AD-30020 283 Modified 39.122 HAMP AD-30021 284 Modified 0.308 HAMP AD-11441 285 Modified 0.042 0.135 0.027 HAMP AD-11458 285 Modified 0.358 HAMP AD-45729 288 Modified 0.068 0.016 HAMP AD-48208 288 Modified 0.012 0.016 HAMP AD-11436 291 Modified 0.054 >10 nM HAMP AD-11453 291 Modified 0.134 HAMP AD-29971 291 Modified 0.108 HAMP AD-29972 294 Modified 0.154 HAMP AD-29974 300 Modified 0.137 HAMP AD-29975 301 Modified 1.392 HAMP AD-45081 309 Modified >10 nM HAMP AD-45087 310 Modified >10 nM HAMP AD-45093 313 Modified >10 nM HAMP AD-29979 352 Modified >10 nM HAMP AD-45750 352 Modified 1.558 HAMP AD-45755 352 Modified 0.296 HAMP AD-45725 355 Modified >10 nM HAMP AD-29981 357 Modified >10 nM HAMP AD-45761 359 Modified >10 nM HAMP AD-45377 364 Modified >10 nM HAMP AD-29982 365 Modified 1.723 HAMP AD-29983 366 Modified >10 nM HAMP AD-47099 370 Unmodified 0.017 0.081 HAMP AD-47114 376 Unmodified 0.008 0.036 HAMP AD-48214 376 Modified 0.008 1.575 HAMP AD-47119 379 Unmodified 0.004 0.040 HAMP AD-47088 381 Unmodified 0.007 >10 nM HAMP AD-11442 382 Modified 0.028 0.010 HAMP AD-11459 382 Unmodified 0.038 0.045 HAMP AD-45062 382 Modified 0.088 0.030 HAMP AD-47094 382 Unmodified 0.005 0.039 HAMP AD-48141 382 Modified 0.004 0.023 HAMP AD-48147 382 Modified 0.007 0.008 HAMP AD-48154 382 Modified 0.006 0.019 HAMP AD-48189 382 Modified 0.005 0.145 HAMP AD-48195 382 Modified 0.011 0.009 HAMP AD-48196 382 Modified 0.017 0.031 HAMP AD-48201 382 Modified 0.007 0.009 HAMP AD-48205 382 Modified 0.014 0.022 HAMP AD-48207 382 Modified 0.007 0.017 HAMP AD-48213 382 Modified 0.009 0.027 HAMP AD-48216 382 Modified 0.014 0.035 HAMP AD-47100 383 Unmodified 0.007 0.172 HAMP AD-48217 385 Modified 0.028 0.021 HAMP AD-47105 396 Unmodified 0.005 >10 nM HAMP AD-48209 396 Modified 0.013 >10 nM HAMP AD-47115 399 Unmodified 0.007 >10 nM HAMP AD-47030 402 Modified 0.015 >10 nM HAMP AD-47120 402 Unmodified 0.006 >10 nM HAMP AD-46993 403 Modified 0.015 >10 nM HAMP AD-47083 403 Unmodified 0.007 >10 nM HAMP AD-46999 407 Modified 0.011 >10 nM

Tables 10A and 10B: Secondary Target Sequences

TABLE 10A Start SEQ Antisense SEQ Target Duplex ID Position Sense Name Sense Sequence ID NO Name Antisense Sequence ID NO HFE2 AD-47391  177 A-98855.1 AGAGuAGGGAAucAu A-98856.1 AGCcAUGAUUCCCuACUC GGcudTsdT UdTsdT HFE2 AD-47397  193 A-98857.1 GcuGGAGAAuuGGAu A-98858.1 UGCuAUCcAAUUCUCcAG AGcAdTsdT CdTsdT HFE2 AD-47403  195 A-98859.1 uGGAGAAuuGGAuAG A-98860.1 UCUGCuAUCcAAUUCUCc cAGAdTsdT AdTsdT HFE2 AD-47409  199 A-98861.1 GAAuuGGAuAGcAGA A-98862.1 UuACUCUGCuAUCcAAUU GuAAdTsdT CdTsdT HFE2 AD-47415  200 A-98863.1 AAuuGGAuAGcAGAG A-98864.1 AUuACUCUGCuAUCcAAU uAAudTsdT UdTsdT HFE2 AD-47421  206 A-98865.1 AuAGcAGAGuAAuGu A-98866.1 UcAAAcAUuACUCUGCuA uuGAdTsdT UdTsdT HFE2 AD-47427  211 A-98867.1 AGAGuAAuGuuuGAc A-98868.1 AGAGGUcAAAcAUuACUC cucudTsdT UdTsdT HFE2 AD-47433  244 A-98869.1 ucAuAuuuAAGAAcA A-98870.1 UGcAUGUUCUuAAAuAUG uGcAdTsdT AdTsdT HFE2 AD-47392  257 A-98871.1 cAuGcAGGAAuGcAu A-98872.1 AUcAAUGcAUUCCUGcAU uGAudTsdT GdTsdT HFE2 AD-47398  261 A-98873.1 cAGGAAuGcAuuGAu A-98874.1 UCUGAUcAAUGcAUUCCU cAGAdTsdT GdTsdT HFE2 AD-47404  290 A-98875.1 GGcuGAGGuGGAuAA A-98876.1 AAGAUuAUCcACCUcAGC ucuudTsdT CdTsdT HFE2 AD-47410  360 A-98877.1 uccAGuuuGucGAuu A-98878.1 UUUGAAUCGAcAAACUGG cAAAdTsdT AdTsdT HFE2 AD-47416  367 A-98879.1 uGucGAuucAAAcuG A-98880.1 UuAGcAGUUUGAAUCGAc cuAAdTsdT AdTsdT HFE2 AD-47422  404 A-98881.1 GAuccAAGcuGccuA A-98882.1 AAUGuAGGcAGCUUGGAU cAuudTsdT CdTsdT HFE2 AD-47428  415 A-98883.1 ccuAcAuuGGcAcAA A-98884.1 AuAGUUGUGCcAAUGuAG cuAudTsdT GdTsdT HFE2 AD-47434  417 A-98885.1 uAcAuuGGcAcAAcu A-98886.1 UuAuAGUUGUGCcAAUGu AuAAdTsdT AdTsdT HFE2 AD-47393  472 A-98887.1 ucAAGGuAGcAGAGG A-98888.1 AcAUCCUCUGCuACCUUG AuGudTsdT AdTsdT HFE2 AD-47399  585 A-98889.1 GGAGcuAuAAccAuu A-98890.1 uAUcAAUGGUuAuAGCUC GAuAdTsdT CdTsdT HFE2 AD-47405  587 A-98891.1 AGcuAuAAccAuuGA A-98892.1 AGuAUcAAUGGUuAuAGC uAcudTsdT UdTsdT HFE2 AD-47417  638 A-98895.1 GGAAGAuGcuuAcuu A-98896.1 AUGGAAGuAAGcAUCUUC ccAudTsdT CdTsdT HFE2 AD-47423  642 A-98897.1 GAuGcuuAcuuccAu A-98898.1 AGGAAUGGAAGuAAGcAU uccudTsdT CdTsdT HFE2 AD-47429  646 A-98899.1 cuuAcuuccAuuccu A-98900.1 AcAcAGGAAUGGAAGuAA GuGudTsdT GdTsdT HFE2 AD-47435  656 A-98901.1 uuccuGuGucuuuGA A-98902.1 AAcAUcAAAGAcAcAGGA uGuudTsdT AdTsdT HFE2 AD-47394  657 A-98903.1 uccuGuGucuuuGAu A-98904.1 AAAcAUcAAAGAcAcAGG GuuudTsdT AdTsdT HFE2 AD-47400  678 A-98905.1 AuuucuGGuGAuccc A-98906.1 AGUUGGGAUcACcAGAAA AAcudTsdT UdTsdT HFE2 AD-47406 1121 A-98907.1 ccAuuuAcuGcAGAu A-98908.1 UGAAAUCUGcAGuAAAUG uucAdTsdT GdTsdT HFE2 AD-47412 1151 A-98909.1 uuAGAGGucAuGAAG A-98910.1 AAACCUUcAUGACCUCuA GuuudTsdT AdTsdT HFE2 AD-47418 1152 A-98911.1 uAGAGGucAuGAAGG A-98912.1 AAAACCUUcAUGACCUCu uuuudTsdT AdTsdT HFE2 AD-47424 1203 A-98913.1 uuAAGAGGcAAGAGc A-98914.1 UUcAGCUCUUGCCUCUuA uGAAdTsdT AdTsdT HFE2 AD-47430 1228 A-98915.1 AGAcAuGAucAuuAG A-98916.1 AUGGCuAAUGAUcAUGUC ccAudTsdT UdTsdT HFE2 AD-47436 1230 A-98917.1 AcAuGAucAuuAGcc A-98918.1 UuAUGGCuAAUGAUcAUG AuAAdTsdT UdTsdT HFE2 AD-47395 1233 A-98919.1 uGAucAuuAGccAuA A-98920.1 UUCUuAUGGCuAAUGAUc AGAAdTsdT AdTsdT HFE2 AD-47401 1272 A-98921.1 AuuAGGGAAAGAAGu A-98922.1 AuAGACUUCUUUCCCuAA cuAudTsdT UdTsdT HFE2 AD-47407 1273 A-98923.1 uuAGGGAAAGAAGuc A-98924.1 AAuAGACUUCUUUCCCuA uAuudTsdT AdTsdT HFE2 AD-51740 1273 A-107281.4 uuAGGGAAAGAAGuC A-107275.3 AAuAGACUUCUUUCCCuA uAuUdTsdT adTsdT HFE2 AD-51747 1273 A-107280.6 uuAGGGAAAGAAGuc A-107277.2 AAuAGACUuCUuUCcCuA uAuUdTsdT AdTsdT HFE2 AD-51744 1273 A-107281.5 uuAGGGAAAGAAGuC A-107276.3 AAuAGACUuCUuUCCCuA uAuUdTsdT AdTsdT HFE2 AD-51731 1273 A-107280.2 uuAGGGAAAGAAGuc A-107273.2 AAuAGACUUCUuUCCCuA uAuUdTsdT AdTsdT HFE2 AD-51736 1273 A-107281.3 uuAGGGAAAGAAGuC A-107274.3 AAuAGACUUCUUUCcCuA uAuUdTsdT AdTsdT HFE2 AD-51732 1273 A-107281.2 uuAGGGAAAGAAGuC A-107273.3 AAuAGACUUCUuUCCCuA uAuUdTsdT AdTsdT HFE2 AD-51734 1273 A-98923.4 uuAGGGAAAGAAGuc A-107274.1 AAuAGACUUCUUUCcCuA uAuudTsdT AdTsdT HFE2 AD-51748 1273 A-107281.6 uuAGGGAAAGAAGuC A-107277.3 AAuAGACUuCUuUCcCuA uAuUdTsdT AdTsdT HFE2 AD-51735 1273 A-107280.3 uuAGGGAAAGAAGuc A-107274.2 AAuAGACUUCUUUCcCuA uAuUdTsdT AdTsdT HFE2 AD-51749 1273 A-107282.6 uuAgGGAAAGAAGuC A-107277.4 AAuAGACUuCUuUCcCuA uAuUdTsdT AdTsdT HFE2 AD-51752 1273 A-107281.7 uuAGGGAAAGAAGuC A-107278.3 AAuAGACUuCUuUCcCuA uAuUdTsdT adTsdT HFE2 AD-51738 1273 A-98923.5 uuAGGGAAAGAAGuc A-107275.1 AAuAGACUUCUUUCCCuA uAuudTsdT adTsdT HFE2 AD-51730 1273 A-98923.3 uuAGGGAAAGAAGuc A-107273.1 AAuAGACUUCUuUCCCuA uAuudTsdT AdTsdT HFE2 AD-51745 1273 A-107282.5 uuAgGGAAAGAAGuC A-107276.4 AAuAGACUuCUuUCCCuA uAuUdTsdT AdTsdT HFE2 AD-51737 1273 A-107282.3 uuAgGGAAAGAAGuC A-107274.4 AAuAGACUUCUUUCcCuA uAuUdTsdT AdTsdT HFE2 AD-51743 1273 A-107280.5 uuAGGGAAAGAAGuc A-107276.2 AAuAGACUuCUuUCCCuA uAuUdTsdT AdTsdT HFE2 AD-51751 1273 A-107280.7 uuAGGGAAAGAAGuc A-107278.2 AAuAGACUuCUuUCcCUA uAuUdTsdT adTsdT HFE2 AD-51750 1273 A-98923.8 uuAGGGAAAGAAGuc A-107278.1 AAuAGACUuCUuUCcCuA uAuudTsdT adTsdT HFE2 AD-51741 1273 A-107282.4 uuAgGGAAAGAAGuC A-107275.4 AAuAGACUUCUUUCCCuA uAuUdTsdT adTsdT HFE2 AD-51742 1273 A-98923.6 uuAGGGAAAGAAGuc A-107276.1 AAuAGACUuCUuUCCCuA uAuudTsdT AdTsdT HFE2 AD-51733 1273 A-107082.2 uuAgGGAAAGAAGuC A-107273.4 AAuAGACUUCUuUCCCuA uAuUdTsdT AdTsdT HFE2 AD-51755 1273 A-107280.8 uuAGGGAAAGAAGuc A-107279.2 AAUAGACUuCUuUCcCuA uAuUdTsdT adTsdT HFE2 AD-51756 1273 A-107281.8 uuAGGGAAAGAAGuC A-107279.3 AAUAGACUuCUuUCcCuA uAuUdTsdT adTsdT HFE2 AD-51728 1273 A-107281.1 uuAGGGAAAGAAGuC A-107272.3 AAuAGACUuCUUUCCCuA uAuUdTsdT AdTsdT HFE2 AD-51729 1273 A-107282.1 uuAgGGAAAGAAGuC A-107272.4 AAuAGACUuCUUUCCCuA uAuUdTsdT AdTsdT HFE2 AD-51726 1273 A-98923.2 uuAGGGAAAGAAGuc A-107272.1 AAuAGACUuCUUUCCCuA uAuudTsdT AdTsdT HFE2 AD-51746 1273 A-98923.7 uuAGGGAAAGAAGuc A-107277.1 AAuAGACUuCUuUCcCuA uAuudTsdT AdTsdT HFE2 AD-51757 1273 A-107282.8 uuAgGGAAAGAAGuC A-107279.4 AAUAGACUuCUuUCcCuA uAuUdTsdT adTsdT HFE2 AD-51727 1273 A-107280.1 uuAGGGAAAGAAGuc A-107272.2 AAuAGACUuCUUUCCCuA uAuUdTsdT AdTsdT HFE2 AD-51753 1273 A-107282.7 uuAgGGAAAGAAGuC A-107278.4 AAuAGACUuCUuUCcCuA uAuUdTsdT adTsdT HFE2 AD-51754 1273 A-98923.9 uuAGGGAAAGAAGuc A-107279.1 AAUAGACUuCUuUCcCuA uAuudTsdT adTsdT HFE2 AD-51739 1273 A-107280.4 uuAGGGAAAGAAGuc A-107275.2 AAuAGACUUCUUUCCCuA uAuUdTsdT adTsdT HFE2 AD-47413 1274 A-98925.1 uAGGGAAAGAAGucu A-98926.1 AAAuAGACUUCUUUCCCu AuuudTsdT AdTsdT HFE2 AD-47419 1279 A-98927.1 AAAGAAGucuAuuuG A-98928.1 UcAUcAAAuAGACUUCUU AuGAdTsdT UdTsdT HFE2 AD-47425 1280 A-98929.1 AAGAAGucuAuuuGA A-98930.1 UUcAUcAAAuAGACUUCU uGAAdTsdT UdTsdT HFE2 AD-47431 1303 A-98931.1 uGuGuGuAAGGuAuG A-98932.1 AGAAcAuACCUuAcAcAc uucudTsdT AdTsdT HFE2 AD-47437 1366 A-98933.1 GuGAAGGGAGucucu A-98934.1 AAGcAGAGACUCCCUUcA GcuudTsdT CdTsdT HFE2 AD-47396 1367 A-98935.1 uGAAGGGAGucucuG A-98936.1 AAAGcAGAGACUCCCUUc cuuudTsdT AdTsdT HFE2 AD-47402 1396 A-98937.1 cAcAGGuAGGAcAGA A-98938.1 uACUUCUGUCCuACCUGU AGuAdTsdT GdTsdT HFE2 AD-47408 1397 A-98939.1 AcAGGuAGGAcAGAA A-98940.1 AuACUUCUGUCCuACCUG GuAudTsdT UdTsdT HFE2 AD-47414 1399 A-98941.1 AGGuAGGAcAGAAGu A-98942.1 UGAuACUUCUGUCCuACC AucAdTsdT UdTsdT HFE2 AD-47420 1400 A-98943.1 GGuAGGAcAGAAGuA A-98944.1 AUGAuACUUCUGUCCuAC ucAudTsdT CdTsdT HFE2 AD-47426 1404 A-98945.1 GGAcAGAAGuAucAu A-98946.1 AGGGAUGAuACUUCUGUC cccudTsdT CdTsdT HFE2 AD-47432 1441 A-98947.1 uAuuAAAGcuAcAAA A-98948.1 AGAAUUUGuAGCUUuAAu uucudTsdT AdTsdT It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.

TABLE 10B Start Sense Antisense Target Duplex ID Position Name Sense Sequence Name Antisense Sequence TFR2 AD-47814   64 A-99594.1 uccAGAGAGcGcAAcAAcudTsdT A-99595.1 AGUUGUUGCGCUCUCUGGAdTsdT TFR2 AD-47820   66 A-99596.1 cAGAGAGcGcAAcAAcuGudTsdT A-99597.1 AcAGUUGUUGCGCUCUCUGdTsdT TFR2 AD-47826  239 A-99598.1 cAGGcAGccAAAccucAuudTsdT A-99599.1 AAUGAGGUUUGGCUGCCUGdTsdT TFR2 AD-47819  772 A-99674.1 AGcuGGuGuAcGcccAcuAdTsdT A-99675.1 uAGUGGGCGuAcACcAGCUdTsdT TFR2 AD-47832  884 A-99600.1 ccAGAAGGuGAccAAuGcudTsdT A-99601.1 AGcAUUGGUcACCUUCUGGdTsdT TFR2 AD-47838  886 A-99602.1 AGAAGGuGAccAAuGcucAdTsdT A-99603.1 UGAGcAUUGGUcACCUUCUdTsdT TFR2 AD-47844  915 A-99604.1 GcucAAGGAGuGcucAuAudTsdT A-99605.1 AuAUGAGcACUCCUUGAGCdTsdT TFR2 AD-47849  916 A-99606.1 cucAAGGAGuGcucAuAuAdTsdT A-99607.1 uAuAUGAGcACUCCUUGAGdTsdT TFR2 AD-47854  920 A-99608.1 AGGAGuGcucAuAuAcccAdTsdT A-99609.1 UGGGuAuAUGAGcACUCCUdTsdT TFR2 AD-47815  922 A-99610.1 GAGuGcucAuAuAcccAGAdTsdT A-99611.1 UCUGGGuAuAUGAGcACUCdTsdT TFR2 AD-47825 1004 A-99676.1 AcAuGuGcAccuGGGAAcudTsdT A-99677.1 AGUUCCcAGGUGcAcAUGUdTsdT TFR2 AD-47821 1048 A-99612.1 cuuccuucAAucAAAcccAdTsdT A-99613.1 UGGGUUUGAUUGAAGGAAGdTsdT TFR2 AD-47827 1050 A-99614.1 uccuucAAucAAAcccAGudTsdT A-99615.1 ACUGGGUUUGAUUGAAGGAdTsdT TFR2 AD-47833 1051 A-99616.1 ccuucAAucAAAcccAGuudTsdT A-99617.1 AACUGGGUUUGAUUGAAGGdTsdT TFR2 AD-51696 1051 A-107271.3 ccuucAAucAAAcccAGuUdTsdT A-107257.2 AACUGGGUUUGAuUGAAGGdTsdT TFR2 AD-51708 1051 A-107271.5 ccuucAAucAAAcccAGuUdTsdT A-107259.2 AACUGGGUuUGAuUGAAGGdTsdT TFR2 AD-51700 1051 A-99616.5 ccuucAAucAAAcccAGuudTsdT A-107258.1 AACUGGGUuUGAUUGAAGGdTsdT TFR2 AD-51701 1051 A-99616.13 ccuucAAucAAAcccAGuudTsdT A-107266.1 AACuGGGUuUGAUUGAAGGdTsdT TFR2 AD-51702 1051 A-107271.4 ccuucAAucAAAcccAGuUdTsdT A-107258.2 AACUGGGUuUGAUUGAAGGdTsdT TFR2 AD-51707 1051 A-99616.14 ccuucAAucAAAcccAGuudTsdT A-107267.1 AACuGGGUuUGAuUGAAGGdTsdT TFR2 AD-51694 1051 A-99616.4 ccuucAAucAAAcccAGuudTsdT A-107257.1 AACUGGGUUUGAuUGAAGGdTsdT TFR2 AD-51706 1051 A-99616.6 ccuucAAucAAAcccAGuudTsdT A-107259.1 AACUGGGUuUGAuUGAAGGdTsdT TFR2 AD-51695 1051 A-99616.12 ccuucAAucAAAcccAGuudTsdT A-107265.1 AACuGGGUUUGAuUGAAGGdTsdT TFR2 AD-51713 1051 A-99616.15 ccuucAAucAAAcccAGuudTsdT A-107268.1 AACuGGGUuUGAuuGAAGGdTsdT TFR2 AD-51714 1051 A-107271.6 ccuucAAucAAAcccAGuUdTsdT A-107260.1 AACUGGGUuUGAuuGAAGGdTsdT TFR2 AD-51683 1051 A-99616.10 ccuucAAucAAAcccAGuudTsdT A-107263.1 AACuGGGUUUGAUUGAAGgdTsdT TFR2 AD-51712 1051 A-99616.7 ccuucAAucAAAcccAGuudTsdT A-107260.1 AACUGGGUuUGAuuGAAGGdTsdT TFR2 AD-51720 1051 A-107271.7 ccuucAAucAAAcccAGuUdTsdT A-107261.2 AACUGGGUuUGAuuGAAGgdTsdT TFR2 AD-51719 1051 A-99616.16 ccuucAAucAAAcccAGuudTsdT A-107269.1 AACuGGGUuUGAuuGAAGgdTsdT TFR2 AD-51684 1051 A-107271.1 ccuucAAucAAAcccAGuUdTsdT A-107255.2 AACUGGGUUUGAUUGAAGgdTsdT TFR2 AD-51690 1051 A-107271.2 ccuucAAucAAAcccAGuUdTsdT A-107256.2 AACUGGGUUUGAUuGAAGGdTsdT TFR2 AD-51689 1051 A-99616.11 ccuucAAucAAAcccAGuudTsdT A-107264.1 AACuGGGUUUGAUuGAAGGdTsdT TFR2 AD-51682 1051 A-99616.2 ccuucAAucAAAcccAGuudTsdT A-107255.1 AACUGGGUUUGAUUGAAGgdTsdT TFR2 AD-51688 1051 A-99616.3 ccuucAAucAAAcccAGuudTsdT A-107256.1 AACUGGGUUUGAUuGAAGGdTsdT TFR2 AD-51718 1051 A-99616.8 ccuucAAucAAAcccAGuudTsdT A-107261.1 AACUGGGUuUGAuuGAAGgdTsdT TFR2 AD-51725 1051 A-99616.17 ccuucAAucAAAcccAGuudTsdT A-107270.1 AACuGGGUuUGAUuGAAGgdTsdT TFR2 AD-51724 1051 A-99616.9 ccuucAAucAAAcccAGuudTsdT A-107262.1 AACUGGGUuUGAUuGAAGgdTsdT TFR2 AD-47839 1067 A-99618.1 GuucccuccAGuuGcAucAdTsdT A-99619.1 UGAUGcAACUGGAGGGAACdTsdT TFR2 AD-47845 1068 A-99620.1 uucccuccAGuuGcAucAudTsdT A-99621.1 AUGAUGcAACUGGAGGGAAdTsdT TFR2 AD-47850 1299 A-99622.1 cGcucAGAGccAGAucAcudTsdT A-99623.1 AGUGAUCUGGCUCUGAGCGdTsdT TFR2 AD-47855 1355 A-99624.1 AGGAGcAGcuAAAuccGcudTsdT A-99625.1 AGCGGAUUuAGCUGCUCCUdTsdT TFR2 AD-47816 1441 A-99626.1 cccGcAGAAGucuccucuudTsdT A-99267.1 AAGAGGAGACUUCUGCGGGdTsdT TFR2 AD-47831 1548 A-99678.1 GuGuAcGuGAGccuGGAcAdTsdT A-99679.1 UGUCcAGGCUcACGuAcACdTsdT TFR2 AD-47822 1584 A-99628.1 GAcAAGuuucAuGccAAGAdTsdT A-99629.1 UCUUGGcAUGAAACUUGUCdTsdT TFR2 AD-47828 1612 A-99630.1 uucuGAcAAGucucAuuGAdTsdT A-99631.1 UcAAUGAGACUUGUcAGAAdTsdT TFR2 AD-47834 1614 A-99632.1 cuGAcAAGucucAuuGAGAdTsdT A-99633.1 UCUcAAUGAGACUUGUcAGdTsdT TFR2 AD-47840 1616 A-99634.1 GAcAAGucucAuuGAGAGudTsdT A-99635.1 ACUCUcAAUGAGACUUGUCdTsdT TFR2 AD-47846 1618 A-99636.1 cAAGucucAuuGAGAGuGudTsdT A-99637.1 AcACUCUcAAUGAGACUUGdTsdT TFR2 AD-47851 2140 A-99638.1 AGcGAcuGAcAcGcAuGuAdTsdT A-99639.1 uAcAUGCGUGUcAGUCGCUdTsdT TFR2 AD-47856 2142 A-99640.1 cGAcuGAcAcGcAuGuAcAdTsdT A-99641.1 UGuAcAUGCGUGUcAGUCGdTsdT TFR2 AD-47817 2143 A-99642.1 GAcuGAcAcGcAuGuAcAAdTsdT A-99643.1 UUGuAcAUGCGUGUcAGUCdTsdT TFR2 AD-47823 2146 A-99644.1 uGAcAcGcAuGuAcAAcGudTsdT A-99645.1 ACGUUGuAcAUGCGUGUcAdTsdT TFR2 AD-47837 2151 A-99680.1 cGcAuGuAcAAcGuGcGcAdTsdT A-99681.1 UGCGcACGUUGuAcAUGCGdTsdT TFR2 AD-47843 2152 A-99682.1 GcAuGuAcAAcGuGcGcAudTsdT A-99683.1 AUGCGcACGUUGuAcAUGCdTsdT TFR2 AD-47829 2154 A-99646.1 AuGuAcAAcGuGcGcAuAAdTsdT A-99647.1 UuAUGCGcACGUUGuAcAUdTsdT TFR2 AD-47835 2155 A-99648.1 uGuAcAAcGuGcGcAuAAudTsdT A-99649.1 AUuAUGCGcACGUUGuAcAdTsdT TFR2 AD-47841 2170 A-99650.1 uAAuGcGGGuGGAGuucuAdTsdT A-99651.1 uAGAACUCcACCCGcAUuAdTsdT TFR2 AD-51703 2170 A-99650.6 uAAuGcGGGuGGAGuucuAdTsdT A-107249.1 UAGAACuCcACCCGcAUuAdTsdT TFR2 AD-51710 2170 A-107254.2 uAAuGcGGGuGGAGuUCuAdTsdT A-107246.3 UAGAACUCcACCCGcAUUAdTsdT TFR2 AD-51697 2170 A-99650.5 uAAuGcGGGuGGAGuucuAdTsdT A-107248.1 UAGAACUCcACCCGcAuuadTsdT TFR2 AD-51692 2170 A-107253.3 uAAuGcGGGuGGAGuuCuAdTsdT A-107247.2 UAGAACUCcACCCGcAUuadTsdT TFR2 AD-51685 2170 A-99650.3 uAAuGcGGGuGGAGuucuAdTsdT A-107246.1 UAGAACUCcACCCGcAUUAdTsdT TFR2 AD-51691 2170 A-99650.4 uAAuGcGGGuGGAGuucuAdTsdT A-107247.1 UAGAACUCcACCCGcAUuadTsdT TFR2 AD-51698 2170 A-107253.4 uAAuGcGGGuGGAGuuCuAdTsdT A-107248.2 UAGAACUCcACCCGcAuuadTsdT TFR2 AD-51686 2170 A-107253.2 uAAuGcGGGuGGAGuuCuAdTsdT A-107246.2 UAGAACUCcACCCGcAuuAdTsdT TFR2 AD-51709 2170 A-99650.7 uAAuGcGGGuGGAGuucuAdTsdT A-107250.1 UAGAACuCcACCCGcAuuAdTsdT TFR2 AD-51679 2170 A-99650.2 uAAuGcGGGuGGAGuucuAdTsdT A-107245.1 UAGAACUCcACCCGcAUuAdTsdT TFR2 AD-51705 2170 A-107254.5 uAAuGcGGGuGGAGuUCuAdTsdT A-107249.3 UAGAACuCcACCCGcAUuAdTsdT TFR2 AD-51704 2170 A-107254.1 uAAuGcGGGuGGAGuUCuAdTsdT A-107245.3 UAGAACUCcACCCGcAUuAdTsdT TFR2 AD-51687 2170 A-107253.6 uAAuGcGGGuGGAGuuCuAdTsdT A-107250.2 UAGAACuCcACCCGcAuuAdTsdT TFR2 AD-51681 2170 A-107253.5 uAAuGcGGGuGGAGuuCuAdTsdT A-107249.2 UAGAACuCcACCCGcAUuAdTsdT TFR2 AD-51716 2170 A-107254.3 uAAuGcGGGuGGAGuUCuAdTsdT A-107247.3 UAGAACUCcACCCGcAUuadTsdT TFR2 AD-51693 2170 A-107253.7 uAAuGcGGGuGGAGuuCuAdTsdT A-107251.2 UAGAACuCcACCCGcUAuadTsdT TFR2 AD-51711 2170 A-107254.6 uAAuGcGGGuGGAGuUCuAdTsdT A-107250.3 UAGAACuCcACCCGcAuuAdTsdT TFR2 AD-51699 2170 A-107253.8 uAAuGcGGGuGGAGuuCuAdTsdT A-107252.2 UAGAACuCcACCCGcAuuadTsdT TFR2 AD-51722 2170 A-107254.4 uAAuGcGGGuGGAGuUCuAdTsdT A-107248.3 UAGAACUCcACCCGcAuuadTsdT TFR2 AD-51715 2170 A-99650.8 uAAuGcGGGuGGAGuucuAdTsdT A-107251.1 UAGAACuCcACCCGcAUuadTsdT TFR2 AD-51680 2170 A-107253.1 uAAuGcGGGuGGAGuuCuAdTsdT A-107245.2 UAGAACUCcACCCGcAUuAdTsdT TFR2 AD-51717 2170 A-107254.7 uAAuGcGGGuGGAGuUCuAdTsdT A-107251.3 UAGAACuCcACCCGcAUuadTsdT TFR2 AD-51723 2170 A-107254.8 uAAuGcGGGuGGAGuUCuAdTsdT A-107252.3 UAGAACuCcACCCGcAuuadTsdT TFR2 AD-51721 2170 A-99650.9 uAAuGcGGGuGGAGuucuAdTsdT A-107252.1 UAGAACuCcACCCGcAuuadTsdT TFR2 AD-47847 2178 A-99652.1 GuGGAGuucuAcuuccuuudTsdT A-99653.1 AAAGGAAGuAGAACUCcACdTsdT TFR2 AD-47852 2224 A-99654.1 cGuuccGccAcAucuucAudTsdT A-99655.1 AUGAAGAUGUGGCGGAACGdTsdT TFR2 AD-47857 2425 A-99656.1 GGAAcAuuGAuAAcAAcuudTsdT A-99657.1 AAGUUGUuAUcAAUGUUCCdTsdT TFR2 AD-47818 2602 A-99658.1 cAGcAcAGAuAuccAcAcAdTsdT A-99659.1 UGUGUGGAuAUCUGUGCUGdTsdT TFR2 AD-47824 2656 A-99660.1 GGucAuAcuGucGGuuAAudTsdT A-99661.1 AUuAACCGAcAGuAUGACCdTsdT TFR2 AD-47830 2658 A-99662.1 ucAuAcuGucGGuuAAucAdTsdT A-99663.1 UGAUuAACCGAcAGuAUGAdTsdT TFR2 AD-47836 2660 A-99664.1 AuAcuGucGGuuAAucAGAdTsdT A-99665.1 UCUGAUuAACCGAcAGuAUdTsdT TFR2 AD-47842 2662 A-99666.1 AcuGucGGuuAAucAGAGAdTsdT A-99667.1 UCUCUGAUuAACCGAcAGUdTsdT TFR2 AD-47848 2719 A-99668.1 GGuccuccAuAccuAGAGAdTsdT A-99669.1 UCUCuAGGuAUGGAGGACCdTsdT TFR2 AD-47853 2795 A-99670.1 ucGcuGGcAccAuAGccuudTsdT A-99671.1 AAGGCuAUGGUGCcAGCGAdTsdT TFR2 AD-47858 2802 A-99672.1 cAccAuAGccuuAuGGccAdTsdT A-99673.1 UGGCcAuAAGGCuAUGGUGdTsdT It should be noted that unmodified versions of each on the modified sequences shown are included within the scope of the invention.

Table 11: Secondary Target Single-Dose

TABLE 11 Start 10 nM overall 0.1 nM overall 0.01 nM overall Target Reactivity Duplex Name Position Avg SD Avg SD Avg SD HFE2 Human AD-47391 177 97.5 10.8 111.9 21.2 HFE2 Human AD-47397 193 27.3 4.2 36.9 3.3 HFE2 Human AD-47403 195 31.2 10.0 48.6 7.6 HFE2 Human AD-47409 199 82.3 15.8 89.5 11.4 HFE2 Human AD-47415 200 44.8 5.9 51.1 7.0 HFE2 Human AD-47421 206 27.8 8.1 28.8 0.9 HFE2 Human AD-47427 211 96.4 25.8 79.8 18.2 HFE2 Human AD-47433 244 7.5 1.3 21.2 4.0 HFE2 Human AD-47392 257 8.6 2.0 20.5 8.1 HFE2 Human AD-47398 261 30.0 6.5 45.9 6.4 HFE2 Human AD-47404 290 9.3 2.8 20.5 0.5 HFE2 Human AD-47410 360 28.7 9.8 36.7 1.8 HFE2 Human AD-47416 367 72.3 32.5 79.2 19.3 HFE2 Human AD-47422 404 20.4 2.5 35.4 2.5 HFE2 Human AD-47428 415 66.8 22.5 80.6 11.4 HFE2 Human AD-47434 417 34.7 5.9 28.6 3.4 HFE2 Human AD-47393 472 96.3 9.7 99.8 31.3 HFE2 Human AD-47399 585 10.0 6.6 16.3 0.6 HFE2 Human AD-47405 587 11.3 2.1 14.0 0.4 HFE2 Human AD-47417 638 39.3 2.0 62.6 7.6 HFE2 Human AD-47423 642 109.4 4.1 58.5 0.9 HFE2 Human AD-47429 646 56.0 13.0 76.3 21.8 HFE2 Human AD-47435 656 17.7 1.4 29.3 9.4 HFE2 Human AD-47394 657 8.8 7.3 9.8 6.3 HFE2 Human AD-47400 678 21.2 2.8 25.1 8.4 HFE2 Human AD-47406 1121 12.9 1.4 20.5 1.3 HFE2 Human AD-47412 1151 16.5 5.2 11.8 3.2 HFE2 Human AD-47418 1152 16.0 1.6 8.4 2.2 HFE2 Human AD-47424 1203 9.2 1.6 14.0 2.4 HFE2 Human AD-47430 1228 14.8 2.7 19.2 0.9 HFE2 Human AD-47436 1230 17.9 9.6 19.7 1.4 HFE2 Human AD-47395 1233 15.3 2.1 12.7 2.2 HFE2 Human AD-47401 1272 6.3 1.2 10.5 0.9 HFE2 Human AD-47407 1273 5.6 1.8 5.6 0.8 HFE2 Human AD-51740 1273 5.7 0.0 6.5 0.7 6.3 0.4 HFE2 Human AD-51747 1273 7.1 1.6 6.0 0.1 7.0 0.1 HFE2 Human AD-51744 1273 11.8 5.8 18.4 14.1 7.7 0.0 HFE2 Human AD-51731 1273 6.2 0.7 7.1 0.2 8.1 3.2 HFE2 Human AD-51736 1273 6.3 0.3 7.2 0.7 8.2 0.5 HFE2 Human AD-51732 1273 6.0 1.0 8.2 0.5 8.3 0.6 HFE2 Human AD-51734 1273 6.9 0.3 14.5 13.3 8.4 1.4 HFE2 Human AD-51748 1273 6.6 0.2 7.7 0.9 8.5 1.3 HFE2 Human AD-51735 1273 6.4 1.5 6.3 0.3 8.5 0.7 HFE2 Human AD-51749 1273 6.8 1.0 8.3 0.4 8.7 2.2 HFE2 Human AD-51752 1273 12.7 6.4 10.3 3.6 8.8 1.0 HFE2 Human AD-51738 1273 5.8 0.6 9.2 3.0 8.9 1.4 HFE2 Human AD-51730 1273 7.6 1.7 7.8 1.0 9.3 0.5 HFE2 Human AD-51745 1273 5.8 0.4 6.5 1.6 9.5 1.2 HFE2 Human AD-51737 1273 5.9 0.1 19.8 18.4 9.6 1.3 HFE2 Human AD-51743 1273 6.5 1.6 7.0 1.5 9.9 2.0 HFE2 Human AD-51751 1273 6.4 1.4 7.5 1.6 10.3 1.6 HFE2 Human AD-51750 1273 6.9 0.2 8.8 0.3 10.7 1.0 HFE2 Human AD-51741 1273 6.0 2.1 8.5 1.1 10.8 4.0 HFE2 Human AD-51742 1273 7.0 1.0 6.1 0.2 11.0 0.9 HFE2 Human AD-51733 1273 6.7 1.1 7.2 0.1 11.0 1.3 HFE2 Human AD-51755 1273 6.1 0.8 13.4 6.9 11.2 2.8 HFE2 Human AD-51756 1273 9.8 0.3 8.9 0.4 11.6 0.3 HFE2 Human AD-51728 1273 7.1 0.8 8.2 0.2 11.6 0.6 HFE2 Human AD-51729 1273 6.8 1.2 8.9 0.4 11.7 0.5 HFE2 Human AD-51726 1273 7.1 0.6 9.0 1.0 12.6 2.4 HFE2 Human AD-51746 1273 7.3 1.4 14.9 6.0 12.6 5.5 HFE2 Human AD-51757 1273 9.1 2.0 10.4 0.6 13.1 1.9 HFE2 Human AD-51727 1273 6.7 1.0 8.5 1.1 13.8 0.4 HFE2 Human AD-51753 1273 7.2 0.3 13.6 8.4 14.2 7.8 HFE2 Human AD-51754 1273 6.9 0.4 10.1 1.0 14.7 2.8 HFE2 Human AD-51739 1273 6.1 0.1 8.2 0.1 14.8 8.9 HFE2 Human AD-47413 1274 7.2 0.2 6.4 0.9 HFE2 Human AD-47419 1279 8.6 2.3 10.0 2.2 HFE2 Human AD-47425 1280 14.5 1.0 14.1 0.8 HFE2 Human AD-47431 1303 49.5 0.6 72.2 0.7 HFE2 Human AD-47437 1366 6.4 4.2 11.4 2.4 HFE2 Human AD-47396 1367 4.6 0.1 10.0 0.2 HFE2 Human AD-47402 1396 11.8 0.2 19.9 4.4 HFE2 Human AD-47408 1397 12.0 3.4 13.7 0.2 HFE2 Human AD-47414 1399 5.6 1.5 8.2 0.1 HFE2 Human AD-47420 1400 3.6 1.0 5.7 0.8 HFE2 Human AD-47426 1404 13.7 3.8 27.1 3.1 HFE2 Human AD-47432 1441 3.8 0.0 5.6 1.0 TFR2 Human AD-47814 64 7.8 0.4 16.3 0.1 TFR2 Human AD-47820 66 13.7 2.5 25.1 3.7 TFR2 Human AD-47826 239 13.5 1.8 25.4 4.3 TFR2 Human AD-47819 772 112.4 2.9 102.9 3.8 TFR2 Human AD-47832 884 24.2 1.8 52.4 2.7 TFR2 Human AD-47838 886 23.6 0.4 39.0 1.6 TFR2 Human AD-47844 915 19.5 3.9 40.9 4.5 TFR2 Human AD-47849 916 14.2 6.9 22.8 0.5 TFR2 Human AD-47854 920 69.4 4.2 88.3 0.8 TFR2 Human AD-47815 922 66.3 6.7 71.2 8.8 TFR2 Human AD-47825 1004 23.9 2.9 46.2 3.8 TFR2 Human AD-47821 1048 57.4 15.9 78.5 5.0 TFR2 Human AD-47827 1050 18.9 8.3 37.9 2.9 TFR2 Human AD-47833 1051 8.3 4.3 19.7 5.4 TFR2 Human AD-51696 1051 8.0 2.1 21.1 2.1 27.2 0.5 TFR2 Human AD-51708 1051 8.8 1.2 17.7 0.8 28.5 3.7 TFR2 Human AD-51700 1051 9.3 1.2 19.8 3.7 30.1 5.0 TFR2 Human AD-51701 1051 9.4 0.6 22.3 8.1 30.8 2.7 TFR2 Human AD-51702 1051 8.7 2.1 19.7 0.1 30.9 1.4 TFR2 Human AD-51707 1051 8.1 2.5 19.1 2.6 32.2 8.2 TFR2 Human AD-51694 1051 9.3 1.9 19.3 2.5 38.8 0.0 TFR2 Human AD-51706 1051 8.4 0.3 19.5 1.5 39.9 6.9 TFR2 Human AD-51695 1051 10.1 1.6 19.9 2.4 40.1 4.2 TFR2 Human AD-51713 1051 14.6 1.8 45.3 2.5 59.0 1.6 TFR2 Human AD-51714 1051 22.1 0.1 44.2 1.5 62.8 1.7 TFR2 Human AD-51683 1051 9.7 0.6 36.5 2.4 66.0 2.4 TFR2 Human AD-51712 1051 21.2 2.6 44.1 4.5 67.1 5.9 TFR2 Human AD-51720 1051 34.5 6.1 58.3 10.4 67.4 0.0 TFR2 Human AD-51719 1051 38.6 1.2 57.7 1.2 68.8 3.2 TFR2 Human AD-51684 1051 14.7 3.5 48.3 1.9 69.3 3.5 TFR2 Human AD-51690 1051 19.7 0.0 49.8 0.5 74.1 12.6 TFR2 Human AD-51689 1051 40.5 2.4 53.1 9.7 75.0 6.4 TFR2 Human AD-51682 1051 12.7 1.1 42.3 10.1 75.7 6.0 TFR2 Human AD-51688 1051 34.9 2.9 62.2 6.9 78.1 3.2 TFR2 Human AD-51718 1051 31.6 6.6 53.1 6.5 80.2 1.3 TFR2 Human AD-51725 1051 47.9 5.0 76.1 1.8 83.7 3.2 TFR2 Human AD-51724 1051 52.0 1.2 66.1 32.9 87.8 14.9 TFR2 Human AD-47839 1067 54.0 3.1 71.5 8.4 TFR2 Human AD-47845 1068 105.7 20.1 98.0 3.0 TFR2 Human AD-47850 1299 16.7 4.8 21.3 3.2 TFR2 Human AD-47855 1355 64.6 0.5 66.1 8.0 TFR2 Human AD-47816 1441 10.6 2.6 30.6 6.9 TFR2 Human AD-47831 1548 22.8 0.2 36.6 9.5 TFR2 Human AD-47822 1584 57.2 7.0 72.6 1.6 TFR2 Human AD-47828 1612 38.2 5.9 61.2 9.9 TFR2 Human AD-47834 1614 9.2 3.6 20.1 3.0 TFR2 Human AD-47840 1616 50.1 3.7 55.6 3.8 TFR2 Human AD-47846 1618 75.0 7.9 94.6 4.3 TFR2 Human AD-47851 2140 94.1 0.4 101.3 10.6 TFR2 Human AD-47856 2142 63.3 4.1 60.7 3.1 TFR2 Human AD-47817 2143 50.2 2.7 50.3 6.5 TFR2 Human AD-47823 2146 26.1 2.3 40.9 3.3 TFR2 Human AD-47837 2151 119.5 21.7 89.5 6.9 TFR2 Human AD-47843 2152 20.6 1.7 34.9 7.8 TFR2 Human AD-47829 2154 53.4 4.1 60.3 0.5 TFR2 Human AD-47835 2155 15.5 1.8 18.3 2.4 TFR2 Human AD-47841 2170 26.6 1.5 24.7 2.0 TFR2 Human AD-51703 2170 25.2 2.8 27.9 3.5 23.2 1.1 TFR2 Human AD-51710 2170 22.1 3.4 23.1 0.5 24.0 0.6 TFR2 Human AD-51697 2170 30.9 3.6 25.3 0.9 24.5 0.8 TFR2 Human AD-51692 2170 23.1 1.3 24.6 1.2 24.9 6.4 TFR2 Human AD-51685 2170 24.6 2.2 23.9 0.6 25.6 1.7 TFR2 Human AD-51691 2170 29.1 3.2 21.3 0.2 26.4 3.7 TFR2 Human AD-51698 2170 23.1 2.3 25.8 3.0 26.8 2.8 TFR2 Human AD-51686 2170 20.7 2.5 24.7 0.7 27.5 1.4 TFR2 Human AD-51709 2170 23.1 1.3 25.1 2.7 27.7 2.1 TFR2 Human AD-51679 2170 27.4 2.2 26.4 4.3 28.3 5.1 TFR2 Human AD-51705 2170 27.8 5.3 24.6 2.0 28.8 2.4 TFR2 Human AD-51704 2170 23.9 2.1 26.1 0.5 29.2 4.6 TFR2 Human AD-51687 2170 20.8 3.9 27.7 2.8 29.4 1.0 TFR2 Human AD-51681 2170 30.0 1.8 31.2 1.8 29.5 4.7 TFR2 Human AD-51716 2170 20.0 1.7 25.9 2.2 30.2 1.1 TFR2 Human AD-51693 2170 26.2 0.8 26.1 1.0 30.6 0.6 TFR2 Human AD-51711 2170 20.8 0.5 24.8 3.2 31.3 3.0 TFR2 Human AD-51699 2170 20.9 0.7 27.3 1.5 31.7 5.1 TFR2 Human AD-51722 2170 28.3 3.7 30.0 0.5 32.1 1.2 TFR2 Human AD-51715 2170 22.2 6.1 30.4 0.6 34.6 1.3 TFR2 Human AD-51680 2170 26.4 2.5 26.7 5.4 36.6 2.6 TFR2 Human AD-51717 2170 28.2 6.2 24.6 0.2 37.2 7.7 TFR2 Human AD-53723 2170 25.9 4.0 30.7 4.0 40.7 3.1 TFR2 Human AD-51721 2170 30.7 1.6 28.1 0.9 40.8 0.3 TFR2 Human AD-47847 2178 21.7 2.1 25.1 3.5 TFR2 Human AD-47852 2224 71.4 2.2 66.7 7.1 TFR2 Human AD-47857 2425 37.4 4.8 29.5 5.4 TFR2 Human AD-47818 2602 48.3 4.8 50.8 4.3 TFR2 Human AD-47824 2656 19.9 3.3 25.7 0.1 TFR2 Human AD-47830 2658 25.8 7.7 25.8 6.4 TFR2 Human AD-47836 2660 34.6 0.1 37.4 6.1 TFR2 Human AD-47842 2662 39.2 6.8 26.3 1.1 TFR2 Human AD-47848 2719 76.8 2.2 90.1 9.7 TFR2 Human AD-47853 2795 28.1 6.3 43.7 3.8 TFR2 Human AD-47858 2802 66.9 8.2 73.6 3.4 Data are expressed as percent of control (Mock transfected or 1955).

Table 12: Secondary Target Dose-Response

TABLE 12 Target Reactivity Duplex Name Start Position IC50 (nM) HFE2 HumaWn AD-47394 657 0.004 HFE2 Human AD-47395 1233 0.011 HFE2 Human AD-47407 1273 0.002 HFE2 Human AD-51747 1273 0.001 HFE2 Human AD-51736 1273 0.001 HFE2 Human AD-51734 1273 0.001 HFE2 Human AD-51732 1273 0.002 HFE2 Human AD-51731 1273 0.002 HFE2 Human AD-51744 1273 0.002 HFE2 Human AD-51748 1273 0.002 HFE2 Human AD-51735 1273 0.002 HFE2 Human AD-47407 1273 0.002 HFE2 Human AD-51740 1273 0.003 HFE2 Human AD-47413 1274 0.003 HFE2 Human AD-47425 1280 0.021 HFE2 Human AD-47437 1366 0.015 HFE2 Human AD-47396 1367 0.013 HFE2 Human AD-47414 1399 0.005 HFE2 Human AD-47420 1400 0.010 HFE2 Human AD-47432 1441 0.004 TFR2 Human AD-47814 64 0.012 TFR2 Human AD-47820 66 0.011 TFR2 Human AD-47826 239 0.014 TFR2 Human AD-47849 916 0.067 TFR2 Human AD-47833 1051 0.013 TFR2 Human AD-51701 1051 0.015 TFR2 Human AD-51708 1051 0.017 TFR2 Human AD-51700 1051 0.017 TFR2 Human AD-47833 1051 0.023 TFR2 Human AD-51696 1051 0.024 TFR2 Human AD-47850 1299 0.011 TFR2 Human AD-47834 1614 0.014 TFR2 Human AD-47835 2155 0.023 TFR2 Human AD-47841 2170 0.009 TFR2 Human AD-51710 2170 0.003 TFR2 Human AD-51703 2170 0.005 TFR2 Human AD-51697 2170 0.006 TFR2 Human AD-51692 2170 0.010 TFR2 Human AD-47841 2170 0.024 TFR2 Human AD-47847 2178 0.013

Table 13: TFR2Duplex Sequences

TABLE 13 Start Sense Antisense Target Duplex ID Position Name Sense Sequence Name Antisense Sequence TFR2 AD-52549  64 A-108802.1 uccAGAGAGcGcAAcAAcUdTsdT A-108798.2 AGUUGUUGCGCUCUCuGGAdTsdT TFR2 AD-52550  64 A-108802.5 uccAGAGAGcGcAAcAAcUdTsdT A-108803.2 AGUUGUUGCGCUCUCuGGadTsdT TFR2 AD-52555  64 A-108802.2 UccAGAGAGcGcAAcAAcUdTsdT A-108799.2 AGUUGUUGCGCUCuCuGGAdTsdT TFR2 AD-52556  64 A-108802.6 uccAGAGAGcGcAAcAAcUdTsdT A-108804.2 AGUUGUUGCGCUCuCuGGadTsdT TFR2 AD-52561  64 A-108802.3 uccAGAGAGcGcAAcAAcUdTsdT A-108800.2 AGUUGUUGCGCuCuCuGGAdTsdT TFR2 AD-52562  64 A-108802.7 uccAGAGAGcGcAAcAAcUdTsdT A-108805.2 AGUUGUUGCGCuCuCuGGadTsdT TFR2 AD-52567  64 A-108802.4 uccAGAGAGcGcAAcAAcUdTsdT A-108801.2 AGUUGuUGCGCuCuCuGGAdTsdT TFR2 AD-52568  64 A-108802.8 uccAGAGAGcGcAAcAAcUdTsdT A-108806.2 AGUUGuUGCGCuCuCuGGadTsdT TFR2 AD-52572  64 A-99594.2 uccAGAGAGcGcAAcAAcudTsdT A-108798.1 AGUUGUUGCGCUCUCuGGAdTsdT TFR2 AD-52573  64 A-99594.6 uccAGAGAGcGcAAcAAcudTsdT A-108803.1 AGUUGUUGCGCUCUCuGGadTsdT TFR2 AD-52577  64 A-99594.3 uccAGAGAGcGcAAcAAcudTsdT A-108799.1 AGUUGUUGCGCUCuCuGGAdTsdT TFR2 AD-52578  64 A-99594.7 uccAGAGAGcGcAAcAAcudTsdT A-108804.1 AGUUGUUGCGCUCuCuGGadTsdT TFR2 AD-52582  64 A-99594.4 uccAGAGAGcGcAAcAAcudTsdT A-108800.1 AGUUGUUGCGCuCuCuGGAdTsdT TFR2 AD-52583  64 A-99594.8 uccAGAGAGcGcAAcAAcudTsdT A-108805.1 AGUUGUUGCGCuCuCuGGadTsdT TFR2 AD-52587  64 A-99594.5 uccAGAGAGcGcAAcAAcudTsdT A-108801.1 AGUUGuUGCGCuCuCuGGAdTsdT TFR2 AD-52588  64 A-99594.9 uccAGAGAGcGcAAcAAcudTsdT A-108806.1 AGUUGuUGCGCuCuCuGGadTsdT TFR2 AD-52551 239 A-108811.1 cAGGcAGccAAAccucAuUdTsdT A-108810.2 AAUGAGGUuUGGCuGcCuGdTsdT TFR2 AD-52552 239 A-108811.3 cAGGcAGccAAAccucAuUdTsdT A-108816.1 AAuGAGGUuUGGCUGCcugdTsdT TFR2 AD-52557 239 A-108812.1 cAGGcAGccAAAccuCAuUdTsdT A-108810.3 AAUGAGGUuUGGCuGcCuGdTsdT TFR2 AD-52558 239 A-108812.3 cAGGcAGccAAAccuCAuUdTsdT A-108816.2 AAuGAGGUuUGGCUGcCugdTsdT TFR2 AD-52563 239 A-108813.1 cAGGcAGccAAAcCuCAuUdTsdT A-108810.4 AAUGAGGUuUGGCuGcCuGdTsdT TFR2 AD-52564 239 A-108813.3 cAGGcAGccAAAcCuCAuUdTsdT A-108816.3 AAuGAGGUuUGGCUGcCugdTsdT TFR2 AD-52569 239 A-108814.1 cAGGcAGcCAAAcCuCAuUdTsdT A-108810.5 AAUGAGGUuUGGCuGcCuGdTsdT TFR2 AD-52570 239 A-108814.3 cAGGcAGcCAAAcCuCAuUdTsdT A-108816.4 AAuGAGGUuUGGCUGcCugdTsdT TFR2 AD-52574 239 A-99598.2 cAGGcAGccAAAccucAuudTsdT A-108807.1 AAUGAGGUUUGGCUGCCuGdTsdT TFR2 AD-52575 239 A-108811.2 cAGGcAGccAAAccucAuUdTsdT A-108815.1 AAUGAGGUuUGGCUGcCugdTsdT TFR2 AD-52579 239 A-99598.3 cAGGcAGccAAAccucAuudTsdT A-108808.1 AAUGAGGUUUGGCUGcCuGdTsdT TFR2 AD-52580 239 A-108812.2 cAGGcAGccAAAccuCAuUdTsdT A-108815.2 AAUGAGGUuUGGCUGcCugdTsdT TFR2 AD-52584 239 A-99598.4 cAGGcAGccAAAccucAuudTsdT A-108809.1 AAUGAGGUUUGGCuGcCuGdTsdT TFR2 AD-52585 239 A-108813.2 cAGGcAGccAAAcCuCAuUdTsdT A-108815.3 AAUGAGGUuUGGCUGcCugdTsdT TFR2 AD-52589 239 A-99598.5 cAGGcAGccAAAccucAuudTsdT A-108810.1 AAUGAGGUuUGGCuGcCuGdTsdT TFR2 AD-52590 239 A-108814.2 cAGGcAGcCAAAcCuCAuUdTsdT A-108815.4 AAUGAGGUuUGGCUGcCugdTsdT It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.

Table 14: TFR2 Dose Response

TABLE 14 Target Reactivity Duplex Name Start Position IC50 (nM) TFR2 Human AD-47814 64 0.019 TFR2 Human AD-52549 64 0.034 TFR2 Human AD-52572 64 0.059 TFR2 Human AD-52550 64 0.062 TFR2 Human AD-52573 64 0.102 TFR2 Human AD-52570 239 0.035 TFR2 Human AD-47826 239 0.036 TFR2 Human AD-52590 239 0.038 TFR2 Human AD-52574 239 0.065 TFR2 Human AD-52558 239 0.236

Table 15: SMAD4 Unmodified Duplexes

TABLE 15 Sense Antis Duplex Name Start Target Oligo Name Trans Seq Oligo Name Trans Seq AD-48090.1  481 SMAD4 A-100350.1 AUGCCUGUCUGAGCAUUGU A-100351.1 ACAAUGCUCAGACAGGCAU AD-48091.1  772 SMAD4 A-100366.1 AUGUUAAAUAUUGUCAGUA A-100367.1 UACUGACAAUAUUUAACAU AD-48092.1  817 SMAD4 A-100382.1 UCUGUGUGAAUCCAUAUCA A-100383.1 UGAUAUGGAUUCACACAGA AD-48093.1 1212 SMAD4 A-100398.1 ACUUACCAUCAUAACAGCA A-100399.1 UGCUGUUAUGAUGGUAAGU AD-48094.1 1351 SMAD4 A-100414.1 ACAAUGAGCUUGCAUUCCA A-100415.1 UGGAAUGCAAGCUCAUUGU AD-48095.1 1712 SMAD4 A-100430.1 UGUUCAUAAGAUCUACCCA A-100431.1 UGGGUAGAUCUUAUGAACA AD-48096.1  590 SMAD4 A-100352.1 AAAAGAUGAAUUGGAUUCU A-100353.1 AGAAUCCAAUUCAUCUUUU AD-48097.1  773 SMAD4 A-100368.1 UGUUAAAUAUUGUCAGUAU A-100369.1 AUACUGACAAUAUUUAACA AD-48098.1  819 SMAD4 A-100384.1 UGUGUGAAUCCAUAUCACU A-100385.1 AGUGAUAUGGAUUCACACA AD-48099.1 1232 SMAD4 A-100400.1 UACCACCUGGACUGGAAGU A-100401.1 ACUUCCAGUCCAGGUGGUA AD-48100.1 1362 SMAD4 A-100416.1 GCAUUCCAGCCUCCCAUUU A-100417.1 AAAUGGGAGGCUGGAAUGC AD-48101.1 1713 SMAD4 A-100432.1 GUUCAUAAGAUCUACCCAA A-100433.1 UUGGGUAGAUCUUAUGAAC AD-48102.1  602 SMAD4 A-100354.1 GGAUUCUUUAAUAACAGCU A-100355.1 AGCUGUUAUUAAAGAAUCC AD-48103.1  777 SMAD4 A-100370.1 AAAUAUUGUCAGUAUGCGU A-100371.1 ACGCAUACUGACAAUAUUU AD-48104.1  820 SMAD4 A-100386.1 GUGUGAAUCCAUAUCACUA A-100387.1 UAGUGAUAUGGAUUCACAC AD-48105.1 1238 SMAD4 A-100402.1 CUGGACUGGAAGUAGGACU A-100403.1 AGUCCUACUUCCAGUCCAG AD-48106.1 1367 SMAD4 A-100418.1 CCAGCCUCCCAUUUCCAAU A-100419.1 AUUGGAAAUGGGAGGCUGG AD-48107.1 2816 SMAD4 A-100434.1 UAUUUCUAGGCACAAGGUU A-100435.1 AACCUUGUGCCUAGAAAUA AD-48108.1  608 SMAD4 A-100356.1 UUUAAUAACAGCUAUAACU A-100357.1 AGUUAUAGCUGUUAUUAAA AD-48109.1  778 SMAD4 A-100372.1 AAUAUUGUCAGUAUGCGUU A-100373.1 AACGCAUACUGACAAUAUU AD-48110.1  861 SMAD4 A-100388.1 AUUGAUCUCUCAGGAUUAA A-100389.1 UUAAUCCUGAGAGAUCAAU AD-48111.1 1250 SMAD4 A-100404.1 UAGGACUGCACCAUACACA A-100405.1 UGUGUAUGGUGCAGUCCUA AD-48112.1 1370 SMAD4 A-100420.1 GCCUCCCAUUUCCAAUCAU A-100421.1 AUGAUUGGAAAUGGGAGGC AD-48113.1 2984 SMAD4 A-100436.1 AAUAUUUUGGAAACUGCUA A-100437.1 UAGCAGUUUCCAAAAUAUU AD-48114.1  611 SMAD4 A-100358.1 AAUAACAGCUAUAACUACA A-100359.1 UGUAGUUAUAGCUGUUAUU AD-48115.1  781 SMAD4 A-100374.1 AUUGUCAGUAUGCGUUUGA A-100375.1 UCAAACGCAUACUGACAAU AD-48116.1 1090 SMAD4 A-100390.1 CUGUGGCUUCCACAAGUCA A-100391.1 UGACUUGUGGAAGCCACAG AD-48117.1 1257 SMAD4 A-100406.1 GCACCAUACACACCUAAUU A-100407.1 AAUUAGGUGUGUAUGGUGC AD-48118.1 1601 SMAD4 A-100422.1 GUUGGAAUGUAAAGGUGAA A-100423.1 UUCACCUUUACAUUCCAAC AD-48119.1 3013 SMAD4 A-100438.1 UAAAUACUGUGCAGAAUAA A-100439.1 UUAUUCUGCACAGUAUUUA AD-48120.1  659 SMAD4 A-100360.1 CAUACAGAGAACAUUGGAU A-100361.1 AUCCAAUGUUCUCUGUAUG AD-48121.1  783 SMAD4 A-100376.1 UGUCAGUAUGCGUUUGACU A-100377.1 AGUCAAACGCAUACUGACA AD-48122.1 1137 SMAD4 A-100392.1 AGUGAAGGACUGUUGCAGA A-100393.1 UCUGCAACAGUCCUUCACU AD-48123.1 1262 SMAD4 A-100408.1 AUACACACCUAAUUUGCCU A-100409.1 AGGCAAAUUAGGUGUGUAU AD-48124.1 1633 SMAD4 A-100424.1 UCAGGUGCCUUAGUGACCA A-100425.1 UGGUCACUAAGGCACCUGA AD-48125.1  698 SMAD4 A-100362.1 UCGGAAAGGAUUUCCUCAU A-100363.1 AUGAGGAAAUCCUUUCCGA AD-48126.1  784 SMAD4 A-100378.1 GUCAGUAUGCGUUUGACUU A-100379.1 AAGUCAAACGCAUACUGAC AD-48126.2  784 SMAD4 A-100378.2 GUCAGUAUGCGUUUGACUU A-100379.2 AAGUCAAACGCAUACUGAC AD-48127.1 1207 SMAD4 A-100394.1 CAGCUACUUACCAUCAUAA A-100395.1 UUAUGAUGGUAAGUAGCUG AD-48128.1 1272 SMAD4 A-100410.1 AAUUUGCCUCACCACCAAA A-100411.1 UUUGGUGGUGAGGCAAAUU AD-48129.1 1650 SMAD4 A-100426.1 CACGCGGUCUUUGUCAAGA A-100427.1 UCUGUACAAAGACCGCGUG AD-48130.1  771 SMAD4 A-100364.1 CAUGUUAAAUAUUGUCAGU A-100365.1 AUCGACAAUAUUUAACAUG AD-48131.1  791 SMAD4 A-100380.1 UGCGUUUGACUUAAAAUGU A-100381.1 ACAUUUUAAGUCAAACGCA AD-48132.1 1209 SMAD4 A-100396.1 GCUACUUACCAUCAUAACA A-100397.1 UGUUAUGAUGGUAAGUAGC AD-48133.1 1273 SMAD4 A-100412.1 AUUUGCCUCACCACCAAAA A-100413.1 UUUUGGUGGUGAGGCAAAU AD-48134.1 1652 SMAD4 A-100428.1 CGCGGUCUUUGUACAGAGU A-100429.1 ACUCUGUACAAAGACCGCG Note that an overhang (e.g. TT, dTsdT) can be added to the 3′ end of any duplex.

Table 16: SMAD4 Modified Duplexes

TABLE 16 Sense Antis Target Duplex Name Start Oligo Name Oligo Seq Oligo Name Oligo Seq SMAD4 AD-48090.1  481 A-100350.1 AuGccuGucuGAGcAuuGudTsdT A-100351.1 AcAAUGCUcAGAcAGGcAUdTsdT SMAD4 AD-48091.1  772 A-100366.1 AuGuuAAAuAuuGucAGuAdTsdT A-100367.1 uACUGAcAAuAUUuAAcAUdTsdT SMAD4 AD-48092.1  817 A-100382.1 ucuGuGuGAAuccAuAucAdTsdT A-100383.1 UGAuAUGGAUUcAcAcAGAdTsdT SMAD4 AD-48093.1 1212 A-100398.1 AcuuAccAucAuAAcAGcAdTsdT A-100399.1 UGCUGUuAUGAUGGuAAGUdTsdT SMAD4 AD-48094.1 1351 A-100414.1 AcAAuGAGcuuGcAuuccAdTsdT A-100415.1 UGGAAUGcAAGCUcAUUGUdTsdT SMAD4 AD-48095.1 1712 A-100430.1 uGuucAuAAGAucuAcccAdTsdT A-100431.1 UGGGuAGAUCUuAUGAAcAdTsdT SMAD4 AD-48096.1  590 A-100352.1 AAAAGAuGAAuuGGAuucudTsdT A-100353.1 AGAAUCcAAUUcAUCUUUUdTsdT SMAD4 AD-48097.1  773 A-100368.1 uGuuAAAuAuuGucAGuAudTsdT A-100369.1 AuACUGAcAAuAUUuAAcAdTsdT SMAD4 AD-48098.1  819 A-100384.1 uGuGuGAAuccAuAucAcudTsdT A-100385.1 AGUGAuAUGGAUUcAcAcAdTsdT SMAD4 AD-48099.1 1232 A-100400.1 uAccAccuGGAcuGGAAGudTsdT A-100401.1 ACUUCcAGUCcAGGUGGuAdTsdT SMAD4 AD-48100.1 1362 A-100416.1 GcAuuccAGccucccAuuudTsdT A-100417.1 AAAUGGGAGGCUGGAAUGCdTsdT SMAD4 AD-48101.1 1713 A-100432.1 GuucAuAAGAucuAcccAAdTsdT A-100433.1 UUGGGuAGAUCUuAUGAACdTsdT SMAD4 AD-48102.1  602 A-100354.1 GGAuucuuuAAuAAcAGcudTsdT A-100355.1 AGCUGUuAUuAAAGAAUCCdTsdT SMAD4 AD-48103.1  777 A-100370.1 AAAuAuuGucAGuAuGcGudTsdT A-100371.1 ACGcAuACUGAcAAuAUUUdTsdT SMAD4 AD-48104.1  820 A-100386.1 GuGuGAAuccAuAucAcuAdTsdT A-100387.1 uAGUGAuAUGGAUUcAcACdTsdT SMAD4 AD-48105.1 1238 A-100402.1 cuGGAcuGGAAGuAGGAcudTsdT A-100403.1 AGUCCuACUUCcAGUCcAGdTsdT SMAD4 AD-48106.1 1367 A-100418.1 ccAGccucccAuuuccAAudTsdT A-100419.1 AUUGGAAAUGGGAGGCUGGdTsdT SMAD4 AD-48107.1 2816 A-100434.1 uAuuucuAGGcAcAAGGuudTsdT A-100435.1 AACCUUGUGCCuAGAAAuAdTsdT SMAD4 AD-48108.1  608 A-100356.1 uuuAAuAAcAGcuAuAAcudTsdT A-100357.1 AGUuAuAGCUGUuAUuAAAdTsdT SMAD4 AD-48109.1  778 A-100372.1 AAuAuuGucAGuAuGcGuudTsdT A-100373.1 AACGcAuACUGAcAAuAUUdTsdT SMAD4 AD-48110.1  861 A-100388.1 AuuGAucucucAGGAuuAAdTsdT A-100389.1 UuAAUCCUGAGAGAUcAAUdTsdT SMAD4 AD-48111.1 1250 A-100404.1 uAGGAcuGcAccAuAcAcAdTsdT A-100405.1 UGUGuAUGGUGcAGUCCuAdTsdT SMAD4 AD-48112.1 1370 A-100420.1 GccucccAuuuccAAucAudTsdT A-100421.1 AUGAUUGGAAAUGGGAGGCdTsdT SMAD4 AD-48113.1 2984 A-100436.1 AAuAuuuuGGAAAcuGcuAdTsdT A-100437.1 uAGcAGUCCCcAAAAuAUUdTsdT SMAD4 AD-48114.1  611 A-100358.1 AAuAAcAGcuAuAAcuAcAdTsdT A-100359.1 UGuAGUuAuAGCUGUuAUUdTsdT SMAD4 AD-48115.1  781 A-100374.1 AuuGucAGuAuGcGuuuGAdTsdT A-100375.1 UcAAACGcAuACUGAcAAUdTsdT SMAD4 AD-48116.1 1090 A-100390.1 cuGuGGcuuccAcAAGucAdTsdT A-100391.1 UGACUUGUGGAAGCcAcAGdTsdT SMAD4 AD-48117.1 1257 A-100406.1 GcAccAUaCaCaccuAAuudTsdT A-100407.1 AAUuAGGUGUGuAUGGUGCdTsdT SMAD4 AD-48118.1 1601 A-100422.1 GuuGGAAuGuAAAGGuGAAdTsdT A-100423.1 UUcACCUUuAcAUUCcAACdTsdT SMAD4 AD-48119.1 3013 A-100438.1 uAAAuAcuGuGcAGAAuAAdTsdT A-100439.1 UuAUUCUGcAcAGuAUUuAdTsdT SMAD4 AD-48120.1  659 A-100360.1 cAuAcAGAGAAcAuuGGAudTsdT A-100361.1 AUCcAAUGUUCUCUGuAUGdTsdT SMAD4 AD-48121.1  783 A-100376.1 uGucAGuAuGcGuuuGAcudTsdT A-100377.1 AGUcAAACGcAuACUGAcAdTsdT SMAD4 AD-48122.1 1137 A-100392.1 AGuGAAGGAcuGuuGcAGAdTsdT A-100393.1 UCUGcAAcAGUCCUUcACUdTsdT SMAD4 AD-48123.1 1262 A-100408.1 AuAcAcAccuAAuuuGccudTsdT A-100409.1 AGGcAAAUuAGGUGUGuAUdTsdT SMAD4 AD-48124.1 1633 A-100424.1 ucAGGuGccuuAGuGAccAdTsdT A-100425.1 UGGUcACuAAGGcACCUGAdTsdT SMAD4 AD-48125.1  698 A-100362.1 ucGGAAAGGAuuuccucAudTsdT A-100363.1 AUGAGGAAAUCCUUUCCGAdTsdT SMAD4 AD-48126.1  784 A-100378.1 GucAGuAuGcGuuuGAcuudTsdT A-100379.1 AAGUcAAACGcAuACUGACdTsdT SMAD4 AD-48126.2  784 A-100378.2 GucAGuAuGcGuuuGAcuudTsdT A-100379.2 AAGUcAAACGcAuACUGACdTsdT SMAD4 AD-48127.1 1207 A-100394.1 cAGcuAcuuAccAucAuAAdTsdT A-100395.1 UuAUGAUGGuAAGuAGCUGdTsdT SMAD4 AD-48128.1 1272 A-100410.1 AAuuuGccucAccAccAAAdTsdT A-100411.1 UUUGGUGGUGAGGcAAAUUdTsdT SMAD4 AD-48120.1 1650 A-100426.1 cAcGcGGucuuuGuAcAGAdTsdT A-100427.1 UCUGuAcAAAGACCGCGUGdTsdT SMAD4 AD-48130.1  771 A-100364.1 cAuGuuAAAuAuuGucAGudTsdT A-100365.1 ACUGAcAAuAUUuAAcAUGdTsdT SMAD4 AD-48131.1  791 A-100380.1 uGcGuuuGAcuuAAAAuGudTsdT A-100381.1 AcAUUUuAAGUcAAACGcAdTsdT SMAD4 AD-48132.1 1209 A-100396.1 GcuAcuuAccAucAuAAcAdTsdT A-100397.1 UGUuAUGAUGGuAAGuAGCdTsdT SMAD4 AD-48133.1 1273 A-100412.1 AuuuGccucAccAccAAAAdTsdT A-100413.1 UUUUGGUGGUGAGGcAAAUdTsdT SMAD4 AD-48134.1 1652 A-100428.1 cGcGGucuuuGuAcAGAGudTsdT A-100429.1 ACUCUGuAcAAAGACCGCGdTsdT It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.

Table 17: NEO1 Unmodified Duplexes

TABLE 17 Sense Antis Target Duplex Name Start OligoName Trans Seq Oligo Name Trans Seq NEO1 AD-48273.1 4618 A-100622.1 CUCCGAGAGUAGCUAUGAA A-100623.1 UUCAUAGCUACUCUCGGAG NEO1 AD-48287.1  546 A-100564.1 GCUCUUCUGUUAUAUUAAA A-100565.1 UUUAAUAUAACAGAAGAGC NEO1 AD-48274.1 5060 A-100638.1 GAGUGUAGACAUUGGCAUU A-100639.1 AAUGCCAAUGUCUACACUC NEO1 AD-48309.1 4778 A-100634.1 GGAAUUGUACAGAGUACGA A-100635.1 UCGUACUCUGUACAAUUCC NEO1 AD-48309.2 4778 A-100634.2 GGAAUUGUACAGAGUACGA A-100635.2 UCGUACUCUGUACAAUUCC NEO1 AD-48297.1 4674 A-100630.1 GACUAAUGAAGGACCUAAA A-100631.1 UUUAGGUCCUUCAUUAGUC NEO1 AD-48296.1 4495 A-100614.1 GAACCAUCACAUUCACUCA A-100615.1 UGAGUGAAUGUGAUGGUUC NEO1 AD-48280.1 5062 A-100640.1 GUGUAGACAUUGGCAUUUA A-100641.1 UAAAUGCCAAUGUCUACAC NEO1 AD-48275.1  535 A-100560.1 CUCAGUUAGAGGCUCUUCU A-100561.1 AGAAGAGCCUCUAACUGAG NEO1 AD-48276.1 1283 A-100576.1 GAUGAUGCUGGGACUUAUU A-100577.1 AAUAAGUCCCAGCAUCAUC NEO1 AD-48269.1  533 A-100558.1 CUCUCAGUUAGAGGCUCUU A-100559.1 AAGAGCCUCUAACUGAGAG NEO1 AD-48286.1 5069 A-100642.1 CAUUGGCAUUUAUGUACAA A-100643.1 UUGUACAUAAAUGCCAAUG NEO1 AD-48299.1  791 A-100568.1 GCAGGUCUUCCAAGAUUUA A-100569.1 UAAAUCUUGGAAGACCUGC NEO1 AD-48295.1 2602 A-100598.1 CCUAGAUGAAACUCGUGUU A-100599.1 AACACGAGUUUCAUCUAGG NEO1 AD-48292.1 5329 A-100644.1 GCAUUGCUGUUUGUAAGCU A-100645.1 AGCUUACAAACAGCAAUGC NEO1 AD-48293.1  686 A-100566.1 GUGGUGCAUUCCAAACACA A-100567.1 UGUGUUUGGAAUGCACCAC NEO1 AD-48288.1 1535 A-100580.1 GUUUUGGGUCUGGUGAAAU A-100581.1 AUUUCACCAGACCCAAAAC NEO1 AD-48307.1 4066 A-100602.1 GCCUGUGAUUAGUGCCCAU A-100603.1 AUGGGCACUAAUCACAGGC NEO1 AD-48270.1 1282 A-100574.1 GGAUGAUGCUGGGACUUAU A-100575.1 AUAAGUCCCAGCAUCAUCC NEO1 AD-48300.1 1949 A-100584.1 GCUCAAAAUAAGCAUGGCU A-100585.1 AGCCAUGCUUAUUUUGAGC NEO1 AD-48306.1 2227 A-100586.1 CCGAGUGGUGGCCUACAAU A-100578.1 AUUGUAGGCCACCACUCGG NEO1 AD-48315.1 5059 A-100636.1 GGAGUGUAGACAUUGGCAU A-100637.1 AUGCCAAUGUCUACACUCC NEO1 AD-48291.1 4673 A-100628.1 GGACUAAUGAAGGACCUAA A-100629.1 UUAGGUCCUUCAUUAGUCC NEO1 AD-48272.1 4096 A-100606.1 CCUCGAUAACCCUCACCAU A-100607.1 AUGGUGAGGGUUAUCGAGG NEO1 AD-48271.1 2273 A-100590.1 GAUGUUGCUGUUCGAACAU A-100591.1 AUGUUCGAACAGCAACAUC NEO1 AD-48294.1 1540 A-100582.1 GGGUCUGGUGAAAUCAGAU A-100583.1 AUCUGAUUUCACCAGACCC NEO1 AD-48278.1 4123 A-100608.1 CUCCAGCAGCCUCGCUUCU A-100609.1 AGAAGCGAGGCUGCUGGAG NEO1 AD-48277.1 2312 A-100592.1 GCUCCUCAGAAUCUGUCCU A-100593.1 AGGACAGAUUCUGAGGAGC NEO1 AD-48313.1 4086 A-100604.1 CCAUCCAUUCCCUCGAUAA A-100605.1 UUAUCGAGGGAAUGGAUGG NEO1 AD-48289.1 2484 A-100596.1 CUCAGCUGAUUGAAGGUCU A-100597.1 AGACCUUCAAUCAGCUGAG NEO1 AD-48290.1 4179 A-100612.1 GGCCCAUUGGCACAUCCAU A-100613.1 AUGGAUGUGCCAAUGGGCC NEO1 AD-48284.1 4174 A-100610.1 CCCAUGGCCCAUUGGCACA A-100611.1 UGUGCCAAUGGGCCAUGGG NEO1 AD-48298.1 6731 A-100646.1 GUACCUGGAUACUGCCACA A-100647.1 UGUGGCAGUAUCCAGGUAC NEO1 AD-48311.1  852 A-100572.1 CAAUUCUGAAUUGUGAAGU A-100573.1 ACUUCACAAUUCAGAAUUG NEO1 AD-48285.1 4664 A-100626.1 CACCUGGAAGGACUAAUGA A-100627.1 UCAUUAGUCCUUCCAGGUG NEO1 AD-48282.1 1448 A-100578.1 CCAACUCCAACUGUGAAGU A-100579.1 ACUUCACAGUUGGAGUUGG NEO1 AD-48302.1 4542 A-100616.1 GAAGGAGCCGGCCUCCUAU A-100617.1 AUAGGAGGCCGGCUCCUUC NEO1 AD-48303.1 4767 A-100632.1 CUUGAAAACAAGGAAUUGU A-100633.1 ACAAUUCCUUGUUUUCAAG NEO1 AD-48279.1 4629 A-100624.1 GCUAUGAACCAGAUGAGCU A-100625.1 AGCUCAUCUGGUUCAUAGC NEO1 AD-48301.1 3361 A-100600.1 GAUACAUGACUGGGUUAUU A-100601.1 AAUAACCCAGUCAUGUAUC NEO1 AD-48314.1 4613 A-100620.1 GAAGACUCCGAGAGUAGCU A-100621.1 AGCUACUCUCGGAGUCUUC NEO1 AD-48312.1 2236 A-100588.1 GGCCUACAAUAAACAUGGU A-100589.1 ACCAUGUUUAUUGUAGGCC NEO1 AD-48304.1 7033 A-100648.1 GUACACACUUGUUUGGCUU A-100649.1 AGGCCAAACAAGUGUGUAC NEO1 AD-48310.1 7043 A-100650.1 GUUUGGCCUUUUCUGUAGU A-100651.1 ACUACAGAAAAGGCCAAAC Note that an overhang (e.g. TT, dTsdT) can be added to the 3′ end of any duplex.

Table 18: NEO1 Modified Duplexes

TABLE 18 Sense Antis Duplex Name Target Start Oligo Name Oligo Seq Oligo Name Oligo Seq AD-48273.1 NEO1 4618 A-100622.1 cuccGAGAGuAGcuAuGAAdTsdT A-100623.1 UUcAuAGCuACUCUCGGAGdTsdT AD-48287.1 NEO1  546 A-100564.1 GcucuucuGuuAuAuuAAAdTsdT A-100565.1 UUuAAuAuAAcAGAAGAGCdTsdT AD-48274.1 NEO1 5060 A-100638.1 GAGuGuAGAcAuuGGcAuudTsdT A-100639.1 AAUGCcAAUGUCuAcACUCdTsdT AD-48309.1 NEO1 4778 A-100634.1 GGAAuuGuAcAGAGuAcGAdTsdT A-100635.1 UCGuACUCUGuAcAAUUCCdTsdT AD-48309.2 NEO1 4778 A-100634.2 GGAAuuGuAcAGAGuAcGAdTsdT A-100635.2 UCGuACUCUGuAcAAUUCCdTsdT AD-48297.1 NEO1 4674 A-100630.1 GAcuAAuGAAGGAccuAAAdTsdT A-100631.1 UUuAGGUCCUUcAUuAGUCdTsdT AD-48296.1 NEO1 4495 A-100614.1 GAAccAucAcAuucAcucAdTsdT A-100615.1 UGAGUGAAUGUGAUGGUUCdTsdT AD-48280.1 NEO1 5062 A-100640.1 GuGuAGAcAuuGGcAuuuAdTsdT A-100641.1 uAAAUGCcAAUGUCuAcACdTsdT AD-48275.1 NEO1  535 A-100560.1 cucAGuuAGAGGcucuucudTsdT A-100561.1 AGAAGAGCCUCuAACUGAGdTsdT AD-48276.1 NEO1 1283 A-100576.1 GAuGAuGcuGGGAcuuAuudTsdT A-100577.1 AAuAAGUCCcAGcAUcAUCdTsdT AD-48269.1 NEO1  533 A-100558.1 cucucAGuuAGAGGcucuudTsdT A-100559.1 AAGAGCCUCuAACUGAGAGdTsdT AD-48286.1 NEO1 5069 A-100642.1 cAuuGGcAuuuAuGuAcAAdTsdT A-100643.1 UUGuAcAuAAAUGCcAAUGdTsdT AD-48299.1 NEO1  791 A-100568.1 GcAGGucuuccAAGAuuuAdTsdT A-100569.1 uAAAUCUUGGAAGACCUGCdTsdT AD-48295.1 NEO1 2602 A-100598.1 ccuAGAuGAAAcucGuGuudTsdT A-100599.1 AAcACGAGUUUcAUCuAGGdTsdT AD-48292.1 NEO1 5329 A-100644.1 GcAuuGcuGuuuGuAAGcudTsdT A-100645.1 AGCUuAcAAAcAGcAAUGCdTsdT AD-48293.1 NEO1  686 A-100566.1 GuGGuGcAuuccAAAcAcAdTsdT A-100567.1 UGUGUUUGGAAUGcACcACdTsdT AD-48288.1 NEO1 1535 A-100580.1 GuuuuGGGucuGGuGAAAudTsdT A-100581.1 AUUUcACcAGACCcAAAACdTsdT AD-48307.1 NEO1 4066 A-100602.1 GccuGuGAuuAGuGcccAudTsdT A-100603.1 AUGGGcACuAAUcAcAGGCdTsdT AD-48270.1 NEO1 1282 A-100574.1 GGAuGAuGcuGGGAcuuAudTsdT A-100575.1 AuAAGUCCcAGcAUcAUCCdTsdT AD-48300.1 NEO1 1949 A-100584.1 GcucAAAAuAAGcAuGGcudTsdT A-100585.1 AGCcAUGCUuAUUUUGAGCdTsdT AD-48306.1 NEO1 2227 A-100586.1 ccGAGuGGuGGccuAcAAudTsdT A-100587.1 AUUGuAGGCcACcACUCGGdTsdT AD-48315.1 NEO1 5059 A-100636.1 GGAGuGuAGAcAuuGGcAudTsdT A-100637.1 AUGCcAAUGUCuAcACUCCdTsdT AD-48291.1 NEO1 4673 A-100628.1 GGAcuAAuGAAGGAccuAAdTsdT A-100629.1 UuAGGUCCUUcAUuAGUCCdTsdT AD-48272.1 NEO1 4096 A-100606.1 ccucGAuAAcccucAccAudTsdT A-100607.1 AUGGUGAGGGUuAUCGAGGdTsdT AD-48271.1 NEO1 2273 A-100590.1 GAuGuuGcuGuucGAAcAudTsdT A-100591.1 AUGUUCGAAcAGcAAcAUCdTsdT AD-48294.1 NEO1 1540 A-100582.1 GGGucuGGuGAAAucAGAudTsdT A-100583.1 AUCUGAUUUcACcAGACCCdTsdT AD-48278.1 NEO1 4123 A-100608.1 cuccAGcAGccucGcuucudTsdT A-100609.1 AGAAGCGAGGCUGCUGGAGdTsdT AD-48277.1 NEO1 2312 A-100592.1 GcuccucAGAAucuGuccudTsdT A-100593.1 AGGAcAGAUUCUGAGGAGCdTsdT AD-48313.1 NEO1 4086 A-100604.1 ccAuccAuucccucGAuAAdTsdT A-100605.1 UuAUCGAGGGAAUGGAUGGdTsdT AD-48289.1 NEO1 2484 A-100596.1 cucAGcuGAuuGAAGGucudTsdT A-100597.1 AGACCUUcAAUcAGCUGAGdTsdT AD-48290.1 NEO1 4179 A-100612.1 GGcccAuuGGcAcAuccAudTsdT A-100613.1 AUGGAUGUGCcAAUGGGCCdTsdT AD-48284.1 NEO1 4174 A-100610.1 cccAuGGcccAuuGGcAcAdTsdT A-100611.1 UGUGCcAAUGGGCcAUGGGdTsdT AD-48298.1 NEO1 6731 A-100646.1 GuAccuGGAuAcuGccAcAdTsdT A-100647.1 UGUGGcAGuAUCcAGGuACdTsdT AD-48311.1 NEO1  852 A-100572.1 cAAuucuGAAuuGuGAAGudTsdT A-100573.1 ACUUcAcAAUUcAGAAUUGdTsdT AD-48285.1 NEO1 4664 A-100626.1 cAccuGGAAGGAcuAAuGAdTsdT A-100627.1 UcAUuAGUCCUUCcAGGUGdTsdT AD-48282.1 NEO1 1448 A-100578.1 ccAAcuccAAcuGuGAAGudTsdT A-100579.1 ACUUcAcAGUUGGAGUUGGdTsdT AD-48302.1 NEO1 4542 A-100616.1 GAAGGAGccGGccuccuAudTsdT A-100617.1 AuAGGAGGCCGGCUCCUUCdTsdT AD-48303.1 NEO1 4767 A-100632.1 cuuGAAAAcAAGGAAuuGudTsdT A-100633.1 AcAAUUCCUUGUUUUcAAGdTsdT AD-48279.1 NEO1 4629 A-100624.1 GcuAuGAAccAGAuGAGcudTsdT A-100625.1 AGCUcAUCUGGUUcAuAGCdTsdT AD-48301.1 NEO1 3361 A-100600.1 GAuAcAuGAcuGGGuuAuudTsdT A-100601.1 AAuAACCcAGUcAUGuAUCdTsdT AD-48314.1 NEO1 4613 A-100620.1 GAAGAcuccGAGAGuAGcudTsdT A-100621.1 AGCuACUCUCGGAGUCUUCdTsdT AD-48312.1 NEO1 2236 A-100588.1 GGccuAcAAuAAAcAuGGudTsdT A-100589.1 ACcAUGUUuAUUGuAGGCCdTsdT AD-48304.1 NEO1 7033 A-100648.1 GuAcAcAcuuGuuuGGccudTsdT A-100649.1 AGGCcAAAcAAGUGUGuACdTsdT AD-48310.1 NEO1 7043 A-100650.1 GuuuGGccuuuucuGuAGudTsdT A-100651.1 ACuAcAGAAAAGGCcAAACdTsdT It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention.

Table 19: SMAD4 Percent Inhibition

TABLE 19 0.1 nM (% 10 nM (% message message remaining) remaning) Target ID Avg SD Avg SD SMAD4 AD-48090 93.6 4.6 54.6 5.6 SMAD4 AD-48091 98.0 5.0 60.8 3.3 SMAD4 AD-48092 64.6 0.2 47.8 12.0 SMAD4 AD-48093 96.4 3.5 45.0 8.0 SMAD4 AD-48094 41.3 0.4 16.3 5.4 SMAD4 AD-48095 64.4 9.1 30.0 0.5 SMAD4 AD-48096 70.5 1.8 44.3 0.7 SMAD4 AD-48097 19.6 2.5 10.0 1.6 SMAD4 AD-48098 60.6 2.1 29.9 1.8 SMAD4 AD-48099 83.1 5.5 57.2 2.5 SMAD4 AD-48100 73.4 1.6 50.4 1.2 SMAD4 AD-48101 34.8 3.7 23.3 0.9 SMAD4 AD-48102 66.9 3.2 35.5 4.0 SMAD4 AD-48103 43.4 8.9 20.5 1.0 SMAD4 AD-48104 53.5 6.2 20.5 1.5 SMAD4 AD-48105 59.4 0.6 23.8 3.0 SMAD4 AD-48106 68.4 0.3 40.7 0.5 SMAD4 AD-48107 40.9 3.0 26.9 6.6 SMAD4 AD-48108 21.4 4.3 15.2 4.3 SMAD4 AD-48109 19.2 4.1 12.1 5.2 SMAD4 AD-48110 46.1 6.4 28.4 8.1 SMAD4 AD-48111 75.9 5.1 68.4 12.1 SMAD4 AD-48112 75.8 2.0 72.0 10.4 SMAD4 AD-48113 87.4 11.1 72.0 2.7 SMAD4 AD-48114 36.7 3.2 19.2 0.6 SMAD4 AD-48115 35.8 2.8 18.6 1.9 SMAD4 AD-48116 37.1 0.2 13.6 0.9 SMAD4 AD-48117 32.1 0.8 21.1 1.4 SMAD4 AD-48118 26.3 1.1 16.4 5.5 SMAD4 AD-48119 52.1 4.7 38.8 4.5 SMAD4 AD-48120 32.1 1.0 13.9 1.4 SMAD4 AD-48121 24.3 2.3 10.0 0.7 SMAD4 AD-48122 31.4 5.7 14.6 1.7 SMAD4 AD-48123 27.4 1.5 14.6 2.2 SMAD4 AD-48124 76.8 7.0 55.8 1.0 SMAD4 AD-48125 28.7 2.6 12.6 0.9 SMAD4 AD-48126 18.9 1.9 7.4 0.2 SMAD4 AD-48127 67.5 3.7 39.6 4.0 SMAD4 AD-48128 69.8 4.0 44.5 6.1 SMAD4 AD-48129 73.1 3.4 42.6 2.0 SMAD4 AD-48130 18.1 0.1 12.5 0.9 SMAD4 AD-48131 44.4 0.5 17.1 4.1 SMAD4 AD-48132 47.7 0.1 22.6 5.4 SMAD4 AD-48133 57.1 1.8 30.4 10.0 SMAD4 AD-48134 86.3 18.0 42.4 9.2

Table 20: NEO1 Percent Inhibition

TABLE 20 0.1 nM (% 10 nM (% message message remaining) remaining) Target ID Avg SD Avg SD Neo1 AD-48273 8.4 0.7 9.3 3.6 Neo1 AD-48287 8.6 5.5 10.4 2.7 Neo1 AD-48274 11.0 4.3 6.5 2.2 Neo1 AD-48309 11.0 0.6 6.5 0.8 Neo1 AD-48297 12.9 1.6 8.7 2.4 Neo1 AD-48296 14.0 6.9 7.6 0.1 Neo1 AD-48280 15.6 3.7 10.8 7.1 Neo1 AD-48275 17.7 6.9 8.4 3.8 Neo1 AD-48276 17.8 9.8 6.8 2.0 Neo1 AD-48269 18.4 5.5 10.9 4.4 Neo1 AD-48286 21.4 3.8 11.7 2.1 Neo1 AD-48299 22.9 3.0 11.7 3.8 Neo1 AD-48295 36.2 16.3 12.0 0.4 Neo1 AD-48292 44.3 6.8 14.8 2.2 Neo1 AD-48293 44.7 14.1 30.7 1.8 Neo1 AD-48288 46.9 21.9 31.9 5.2 Neo1 AD-48307 50.2 10.1 16.8 3.9 Neo1 AD-48270 54.2 10.6 65.9 42.5 Neo1 AD-48300 54.6 0.1 18.6 1.9 Neo1 AD-48306 56.6 19.5 16.0 2.3 Neo1 AD-48315 57.7 3.5 17.6 8.0 Neo1 AD-48291 60.2 12.0 35.2 6.4 Neo1 AD-48272 61.9 4.1 25.2 3.2 Neo1 AD-48271 62.6 4.7 35.4 6.8 Neo1 AD-48294 62.6 2.1 22.7 11.0 Neo1 AD-48278 62.9 13.8 27.4 1.3 Neo1 AD-48277 63.2 20.4 26.1 2.6 Neo1 AD-48313 68.2 18.7 43.7 2.2 Neo1 AD-48289 70.6 15.3 53.6 12.3 Neo1 AD-48290 73.8 22.6 60.0 3.9 Neo1 AD-48284 74.0 19.2 106.9 43.7 Neo1 AD-48298 76.0 6.9 75.4 19.3 Neo1 AD-48311 77.9 22.6 23.5 11.1 Neo1 AD-48285 81.0 11.5 65.3 14.2 Neo1 AD-48282 82.7 16.3 47.0 15.3 Neo1 AD-48302 83.3 3.1 32.8 6.7 Neo1 AD-48303 85.0 16.3 29.2 7.7 Neo1 AD-48279 90.2 6.2 51.7 14.3 Neo1 AD-48301 91.8 8.5 88.2 11.1 Neo1 AD-48314 96.7 16.7 128.8 37.8 Neo1 AD-48312 107.9 30.0 94.0 27.8 Neo1 AD-48304 111.6 22.3 91.6 33.2 Neo1 AD-48310 118.0 36.4 118.8 29.0

Table 21: BMP6 Duplexes

TABLE 21 SEQ SEQ duplexName sOligoSeq ID NO asOligoSeq ID NO Set AD-47955.1 GcAGAAuuccGcAucuAcAdTsdT UGuAGAUGCGGAAUUCUGCdTsdT humanRhesus AD-47957.1 GAAuAuGGuuGuAAGAGcudTsdT AGCUCUuAcAACcAuAUUCdTsdT humanRhesus AD-47966.1 cucuucAuGcuGGAucuGudTsdT AcAGAUCcAGcAUGAAGAGdTsdT humanRhesus AD-47989.1 GAGuucAAGuucAAcuuAudTsdT AuAAGUUGAACUUGAACUCdTsdT humanRhesus AD-47993.1 cGuGAGuAGuuGuuGGucudTsdT AGACcAAcAACuACUcACGdTsdT humanRhesus AD-47960.1 GGAcGAccAuGAGAGAuAAdTsdT UuAUCUCUcAUGGUCGUCCdTsdT humanRhesus AD-47997.1 ccuAGAuuAcAucuGccuudTsdT AAGGcAGAUGuAAUCuAGGdTsdT humanRhesus AD-47985.1 cAAcAGAGucGuAAucGcudTsdT AGCGAUuACGACUCUGUUGdTsdT humanRhesus AD-47983.1 GucuAucAAAGGuAGAuuudTsdT AAAUCuACCUUUGAuAGACdTsdT humanRhesus AD-47954.1 cccGGAcGAccAuGAGAGAdTsdT UCUCUcAUGGUCGUCCGGGdTsdT humanRhesus AD-47972.1 cucGucAGcGAcAccAcAAdTsdT UUGUGGUGUCGCUGACGAGdTsdT humanRhesus AD-47981.1 ccAcuAAcucGAAAccAGAdTsdT UCUGGUUUCGAGUuAGUGGdTsdT humanRhesus AD-47982.1 GuAAAuGAcGuGAGuAGuudTsdT AACuACUcACGUcAUUuACdTsdT humanRhesus AD-47987.1 GGGGAcAcAcAuucuGccudTsdT AGGcAGAAUGUGUGUCCCCdTsdT humanRhesus AD-47994.1 cGGcuGcAGAAuuccGcAudTsdT AUGCGGAAUUCUGcAGCCGdTsdT humanRhesus AD-47973.1 GccGAcAAcAGAGucGuAAdTsdT UuACGACUCUGUUGUCGGCdTsdT humanRhesus AD-47975.1 GGAuGccAcuAAcucGAAAdTsdT UUUCGAGUuAGUGGcAUCCdTsdT humanRhesus AD-47979.1 ccGAcAAcAGAGucGuAAudTsdT AUuACGACUCUGUUGUCGGdTsdT humanRhesus AD-47996.1 cGuGcuGuGcGccAAcuAAdTsdT UuAGUUGGCGcAcAGcACGdTsdT humanRhesus AD-47968.1 cAAcGcAcAcAuGAAuGcAdTsdT UGcAUUcAUGUGUGCGUUGdTsdT humanRhesus AD-47977.1 cuGucuAucAAAGGuAGAudTsdT AUCuACCUUUGAuAGAcAGdTsdT humanRhesus AD-47995.1 GcGGGucuccAGuGcuucAdTsdT UGAAGcACUGGAGACCCGCdTsdT humanRhesus AD-47959.1 cuGAGuuuGGAuGucuGuAdTsdT uAcAGAcAUCcAAACUcAGdTsdT humanRhesus AD-47962.1 cAGGAAGcAuGAGcuGuAudTsdT AuAcAGCUcAUGCUUCCUGdTsdT humanRhesus AD-47967.1 GGcuGGcuGGAAuuuGAcAdTsdT UGUcAAAUUCcAGCcAGCCdTsdT humanRhesus AD-47986.1 GcAGAccuuGGuucAccuudTsdT AAGGUGAACcAAGGUCUGCdTsdT humanRhesus AD-47988.1 GAcGuGAGuAGuuGuuGGudTsdT ACcAAcAACuACUcACGUCdTsdT humanRhesus AD-47990.1 cAGAGucGuAAucGcucuAdTsdT uAGAGCUGAuACGACUCUGdTsdT humanRhesus AD-47991.1 cAGAccuuGGuucAccuuAdTsdT uAAGGUGAACcAAGGUCUGdTsdT humanRhesus AD-47956.1 GGGucuccAGuGcuucAGAdTsdT UCUGAAGcACUGGAGACCCdTsdT humanRhesus AD-47974.1 GcAcAcAuGAAuGcAAccAdTsdT UGGUUGcAUUcAUGUGUGCdTsdT humanRhesus AD-47976.1 GGuAAAuGAcGuGAGuAGudTsdT ACuACUcACGUcAUUuACCdTsdT humanRhesus AD-47980.1 cAcAcAuGAAuGcAAccAAdTsdT UUGGUUGcAUUcAUGUGUGdTsdT humanRhesus AD-47984.1 cGAcAccAcAAAGAGuucAdTsdT UGAACUCUUUGUGGUGUCGdTsdT humanRhesus AD-47964.1 cucAuuAAuAAuuuGcucAdTsdT UGAGcAAAUuAUuAAUGAGdTsdT humanRhesus AD-47970.1 cAuuAAuAAuuuGcucAcudTsdT AGUGAGcAAAUuAUuAAUGdTsdT humanRhesus AD-47971.1 GuAcuGucuAucAAAGGuAdTsdT uACCUUUGAuAGAcAGuACdTsdT humanRhesus AD-47963.1 cuuGuGGAuGccAcuAAcudTsdT AGUuAGUGGcAUCcAcAAGdTsdT humanRhesus AD-47965.1 GuucAGuAcuGucuAucAAdTsdT UUGAuAGAcAGuACUGAACdTsdT humanRhesus AD-47992.1 cuuGGAuuccuAGAuuAcAdTsdT UGuAAUCuAGGAAUCcAAGdTsdT humanRhesus AD-47998.1 GGucuGuAGcAAGcuGAGudTsdT ACUcAGCUUGCuAcAGACCdTsdT humanRhesus AD-47958.1 GAuuuuAAAGGAccucAuudTsdT AAUGAGGUCCUUuAAAAUCdTsdT humanRhesus AD-47961.1 cAAAcuuuucuuAucAGcAdTsdT UGCUGAuAAGAAAAGUUUGdTsdT humanRhesus AD-47969.1 GuGGAuGccAcuAAcucGAdTsdT UCGAGUuAGUGGcAUCcACdTsdT humanRhesus AD-47978.1 GucAGcGAcAccAcAAAGAdTsdT UCUUUGUGGUGUCGCUGACdTsdT humanRhesus AD-47305.1 ucAuGAGcuuuGuGAAccudTsdT AGGUUcAcAAAGCUcAUGAdTsdT humanRhesus Mouse AD-47325.1 GAGAcGGcccuuAcGAcAAdTsdT UUGUCGuAAGGGCCGUCUCdTsdT humanRhesus Mouse AD-47329.1 AcGGcccuuAcGAcAAGcAdTsdT UGCUUGUCGuAAGGGCCGUdTsdT humanRhesus Mouse AD-47309.1 AAccuGGuGGAGuAcGAcAdTsdT UGUCGuACUCcACcAGGUUdTsdT humanRhesus Mouse AD-47317.1 GcAGAGAcGGcccuuAcGAdTsdT UCGuAAGGGCCGUCUCUGCdTsdT humanRhesus Mouse AD-47313.1 AccuGGuGGAGuAcGAcAAdTsdT UUGUCGuACUCcACcAGGUdTsdT humanRhesus Mouse AD-47321.1 AGAGAcGGcccuuAcGAcAdTsdT UGUCGuAAGGGCCGUCUCUdTsdT humanRhesus Mouse AD-47333.1 ucccAcucAAcGcAcAcAudTsdT AUGUGUGCGUUGAGUGGGAdTsdT humanRhesus Mouse AD-48038.1 ucAAcGAcGcGGAcAuGGudTsdT ACcAUGUCCGCGUCGUUGAdTsdT mouseRat AD-48010.1 GccAucucGGuucuuuAcudTsdT AGuAAAGAACCGAGAUGGCdTsdT mouseRat AD-48042.1 AAuGccAucucGGuucuuudTsdT AAAGAACCGAGAUGGcAUUdTsdT mouseRat AD-48000.1 AAcGAcGcGGAcAuGGucAdTsdT UGACcAUGUCCGCGUCGUUdTsdT mouseRat AD-48004.1 AuGccAucucGGuucuuuAdTsdT uAAAGAACCGAGAUGGcAUdTsdT mouseRat It should be noted that unmodified versions of each of the modified sequences shown are included within the scope of the invention. 

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of hepcidin antimicrobial peptide (HAMP), wherein said dsRNA is selected from the dsRNAs listed in Table 2, 3, 4, or 5 with a start position of 382, 380, 379, or
 385. 2. The dsRNA of claim 1, wherein the dsRNA consists of AD-48141, wherein the sense strand of AD-48141 is GAAcAuAGGucuuGGAAuAdTdT and the antisense strand of AD-48141 is UAuUCcAAGACCuAuGuUCdTdT.
 3. A dsRNA for inhibiting expression of HAMP, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a HAMP mRNA transcript, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 2, 3, 4, or
 5. 4. The dsRNA of claim 3, wherein the region of complementarity is at least 17 nucleotides in length.
 5. The dsRNA of any one of claims 3-4, wherein the region of complementarity is between 19 and 21 nucleotides in length.
 6. The dsRNA of any one of claims 3-5, wherein the region of complementarity is 19 nucleotides in length.
 7. The dsRNA of any one of claims 3-6, wherein the region of complementarity consists of one of the antisense strand sequences of Table 2, 3, 4, or
 5. 8. The dsRNA of any one of claims 3-7, wherein the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 2, 3, 4, or
 5. 9. The dsRNA of any one of claims 3-8, wherein the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 2, 3, 4, or
 5. 10. The dsRNA of any one of claims 3-9, wherein the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 2, 3, 4, or
 5. 11. The dsRNA of any one of claims 3-10, wherein the sense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the antisense strand sequences of Table 2, 3, 4, or
 5. 12. The dsRNA of any one of claims 3-11, wherein the sense strand comprises one of the sense strand sequences of Table 2, 3, 4, or
 5. 13. The dsRNA of any one of claims 3-12, wherein the antisense strand comprises one of the antisense strand sequences of Table 2, 3, 4, or
 5. 14. The dsRNA of any one of claims 3-13, wherein the sense strand comprises one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand comprises one of the antisense strand sequences of Table 2, 3, 4, or
 5. 15. The dsRNA of any one of claims 3-14, wherein the sense strand consists of one of the sense strand sequences of Table 2, 3, 4, or 5 and the antisense strand consists of one of the antisense strand sequences of Table 2, 3, 4, or
 5. 16. The dsRNA of any one of claims 3-15, wherein the dsRNA mediates degradation of HAMP mRNA.
 17. The dsRNA of any one of claims 3 through 16, wherein said dsRNA further comprises at least one modified nucleotide.
 18. The dsRNA of claim 17, wherein at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
 19. The dsRNA of claim 17, wherein said modified nucleotide is chosen from the group consisting of: a 2′-fluoro modified nucleotide, a 2′-fluoro modified nucleoside, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
 20. The dsRNA of any one of claims 3 through 19, wherein each strand is no more than 30 nucleotides in length.
 21. The dsRNA of any one of claims 3 through 20, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.
 22. The dsRNA of any one of claims 3-21, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.
 23. The dsRNA of any one of claims 3-22, wherein each strand comprises a 3′ overhang of 2 nucleotides.
 24. The dsRNA of any one of claims 1 through 23, further comprising a ligand.
 25. The dsRNA of claim 24, wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.
 26. The dsRNA of any one of claims 1 through 25, further comprising an N-Acetyl-Galactosamine (GalNac) conjugate.
 27. The dsRNA of any one of claims 1 through 26, wherein the dsRNA is formulated in a nucleic acid lipid particle formulation.
 28. The dsRNA of claim 27, wherein the nucleic acid lipid particle formulation is selected from Table A.
 29. The dsRNA of claim 27, wherein the nucleic acid lipid particle formulation comprises MC3.
 30. A cell comprising the dsRNA of any one of claims 1 through
 29. 31. A vector encoding at least one strand of the dsRNA of any one of claims 1 through
 29. 32. A cell comprising the vector of claim
 31. 33. A pharmaceutical composition for inhibiting expression of a HAMP gene comprising the dsRNA of any one of claims 1 through
 29. 34. The pharmaceutical composition of claim 33, further comprising a lipid formulation.
 35. The pharmaceutical composition of claim 34, wherein the lipid formulation is a nucleic acid lipid particle formulation.
 36. A dsRNA for inhibiting expression of hemojuvelin (HFE2), wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a HFE2 mRNA transcript, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10A.
 37. The dsRNA of claim 36, wherein the region of complementarity is at least 17 nucleotides in length.
 38. The dsRNA of any one of claims 36-37, wherein the region of complementarity is between 19 and 21 nucleotides in length.
 39. The dsRNA of any one of claims 36-38, wherein the region of complementarity is 19 nucleotides in length.
 40. The dsRNA of any one of claims 36-39, wherein the region of complementarity consists of one of the antisense strand sequences of Table 10A.
 41. The dsRNA of any one of claims 36-40, wherein the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10A.
 42. The dsRNA of any one of claims 36-41, wherein the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10A.
 43. The dsRNA of any one of claims 36-42, wherein the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10A and the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10A.
 44. The dsRNA of any one of claims 36-43, wherein the sense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the sense strand sequences of Table 10A and the antisense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the antisense strand sequences of Table 10A.
 45. The dsRNA of any one of claims 36-44, wherein the sense strand comprises one of the sense strand sequences of Table 10A.
 46. The dsRNA of any one of claims 36-45, wherein the antisense strand comprises one of the antisense strand sequences of Table 10A.
 47. The dsRNA of any one of claims 36-46, wherein the sense strand comprises one of the sense strand sequences of Table 10A and the antisense strand comprises one of the antisense strand sequences of Table 10A.
 48. The dsRNA of any one of claims 36-47, wherein the sense strand consists of one of the sense strand sequences of Table 10A and the antisense strand consists of one of the antisense strand sequences of Table 10A.
 49. The dsRNA of any one of claims 36-48, wherein the dsRNA mediates degradation of HFE2 mRNA.
 50. The dsRNA of any one of claims 36 through 49, wherein said dsRNA further comprises at least one modified nucleotide.
 51. The dsRNA of claim 50, wherein at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
 52. The dsRNA of claim 50, wherein said modified nucleotide is chosen from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
 53. The dsRNA of any one of claims 36 through 52, wherein each strand is no more than 30 nucleotides in length.
 54. The dsRNA of any one of claims 36 through 53, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.
 55. The dsRNA of claim 54, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.
 56. The dsRNA of any one of claims 36 through 55, further comprising a ligand.
 57. The dsRNA of claim 56, wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.
 58. The dsRNA of any one of claims 36 through 57, further comprising a GalNac conjugate.
 59. The dsRNA of any one of claims 36 through 58, wherein the dsRNA is formulated in a nucleic acid lipid particle formulation.
 60. The dsRNA of claim 59, wherein the nucleic acid lipid particle formulation is selected from Table A.
 61. The dsRNA of claim 59, wherein the nucleic acid lipid particle formulation comprises MC3.
 62. A cell comprising the dsRNA of any one of claims 36 through
 61. 63. A vector encoding at least one strand of the dsRNA of any one of claims 36 through
 61. 64. A cell comprising the vector of claim
 63. 65. A pharmaceutical composition for inhibiting expression of a HFE2 gene comprising the dsRNA of any one of claims 36 through
 61. 66. The pharmaceutical composition of claim 65, further comprising a lipid formulation.
 67. The pharmaceutical composition of claim 66, wherein the lipid formulation is a nucleic acid lipid particle formulation.
 68. A dsRNA for inhibiting expression of transferrin receptor 2 (TFR2), wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to a TFR2 mRNA transcript, wherein said dsRNA is selected from the dsRNAs listed in Table 10B or 13 with a start position of 239 or 64; or wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10B or 13; or wherein the dsRNA is AD-52590.
 69. The dsRNA of claim 68, wherein the region of complementarity is at least 17 nucleotides in length.
 70. The dsRNA of any one of claims 68-69, wherein the region of complementarity is between 19 and 21 nucleotides in length.
 71. The dsRNA of any one of claims 68-70, wherein the region of complementarity is 19 nucleotides in length.
 72. The dsRNA of any one of claims 68-71, wherein the region of complementarity consists of one of the antisense strand sequences of Table 10B or
 13. 73. The dsRNA of any one of claims 68-72, wherein the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10B or
 13. 74. The dsRNA of any one of claims 68-73, wherein the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10B or
 13. 75. The dsRNA of any one of claims 68-74, wherein the sense strand comprises 15 or more contiguous nucleotides of one of the sense strand sequences of Table 10B or 13 and the antisense strand comprises 15 or more contiguous nucleotides of one of the antisense strand sequences of Table 10B or
 13. 76. The dsRNA of any one of claims 68-75, wherein the sense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the sense strand sequences of Table 10B or 13 and the antisense strand comprises 16, 17, 18, 19, 20, or more contiguous nucleotides of one of the antisense strand sequences of Table 10B or
 13. 77. The dsRNA of any one of claims 68-76, wherein the sense strand comprises one of the sense strand sequences of Table 10B or
 13. 78. The dsRNA of any one of claims 68-77, wherein the antisense strand comprises one of the antisense strand sequences of Table 10B or
 13. 79. The dsRNA of any one of claims 68-78, wherein the sense strand comprises one of the sense strand sequences of Table 10B or 13 and the antisense strand comprises one of the antisense strand sequences of Table 10B or
 13. 80. The dsRNA of any one of claims 68-79, wherein the sense strand consists of one of the sense strand sequences of Table 10B or 13 and the antisense strand consists of one of the antisense strand sequences of Table 10B or
 13. 81. The dsRNA of any one of claims 68-80, wherein the dsRNA mediates degradation of TFR2 mRNA.
 82. The dsRNA of any one of claims 68 through 81, wherein said dsRNA further comprises at least one modified nucleotide.
 83. The dsRNA of claim 82, wherein at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
 84. The dsRNA of claim 82, wherein said modified nucleotide is chosen from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
 85. The dsRNA of any one of claims 68 through 84, wherein each strand is no more than 30 nucleotides in length.
 86. The dsRNA of any one of claims 68 through 85, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.
 87. The dsRNA of any one of claims 68-86, wherein at least one strand comprises a 3′ overhang of 2 nucleotides.
 88. The dsRNA of any one of claims 68 through 87, further comprising a ligand.
 89. The dsRNA of claim 88, wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.
 90. The dsRNA of any one of claims 68 through 89, further comprising a GalNac conjugate.
 91. The dsRNA of any one of claims 68 through 89, wherein the dsRNA is formulated in a nucleic acid lipid particle formulation.
 92. The dsRNA of claim 91, wherein the nucleic acid lipid particle formulation is selected from Table A.
 93. The dsRNA of claim 91, wherein the nucleic acid lipid particle formulation comprises MC3.
 94. A cell comprising the dsRNA of any one of claims 68 through
 93. 95. A vector encoding at least one strand of the dsRNA of any one of claims 68 through
 93. 96. A cell comprising the vector of claim
 95. 97. A pharmaceutical composition for inhibiting expression of a TFR2 gene comprising the dsRNA of any one of claims 68 through
 93. 98. The pharmaceutical composition of claim 97, further comprising a lipid formulation.
 99. The pharmaceutical composition of claim 98, wherein the lipid formulation is a nucleic acid lipid particle formulation.
 100. A composition comprising a first dsRNA for inhibiting expression of a HAMP gene and a second dsRNA for inhibiting expression of an HFE2 gene, wherein the first dsRNA comprises a first sense strand and an first antisense strand, the first antisense strand comprising a region of complementarity to a HAMP mRNA transcript, wherein the first antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 2, 3, 4, or 5; and wherein the second dsRNA comprises a second sense strand and a second antisense strand, the second antisense strand comprising a region of complementarity to a HFE2 mRNA transcript, wherein the second antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10A.
 101. A composition comprising a first dsRNA for inhibiting expression of a HAMP gene and a second dsRNA for inhibiting expression of an TFR2 gene, wherein said first dsRNA comprises a first sense strand and a first antisense strand, the first antisense strand comprising a region of complementarity to a HAMP mRNA transcript, wherein the first antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 2, 3, 4, or 5; and wherein said second dsRNA comprises a second sense strand and a second antisense strand, the second antisense strand comprising a region of complementarity to a TFR2 mRNA transcript, wherein the second antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10B or
 13. 102. A composition comprising a first dsRNA for inhibiting expression of a TFR2 gene and a second dsRNA for inhibiting expression of a HFE2 gene, wherein said first dsRNA comprises a first sense strand and a first antisense strand, the first antisense strand comprising a region of complementarity to a TFR2 mRNA transcript, wherein the first antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10B or 13; and wherein said second dsRNA comprises a second sense strand and a second antisense strand, the second antisense strand comprising a region of complementarity to a HFE2 mRNA transcript, wherein the second antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense strand sequences listed in Table 10A.
 103. A composition comprising a plurality of dsRNAs selected from the dsRNAs of any one of claims 1 through 29, 36 through 61, and 68 through
 93. 104. A method of inhibiting HAMP expression in a cell, the method comprising: (a) introducing into the cell the dsRNA of any one of claims 1 through 29; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a HAMP gene, thereby inhibiting expression of the HAMP gene in the cell.
 105. The method of claim 104, wherein the HAMP expression is inhibited by at least 30%.
 106. The method of any one of claims 104-105, wherein the HAMP expression is inhibited by at least 80%.
 107. A method of treating a disorder associated with HAMP expression comprising administering to a subject in need of such treatment a therapeutically effective amount of the dsRNA of any of claims 1 through
 29. 108. The method of claim 107, wherein the subject has anemia.
 109. The method of any one of claims 107-108, wherein the subject has refractory anemia.
 110. The method of any one of claims 107-109, wherein the subject has anemia of chronic disease (ACD).
 111. The method of any one of claims 107-110, wherein the subject has iron-restricted erythropoiesis.
 112. The method of any one of claims 107-111, wherein the subject is a human.
 113. The method of any one of claims 107-112, wherein the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
 114. The method of any one of claims 107-113, wherein the dsRNA is lipid formulated.
 115. The method of any one of claims 107-114, wherein the dsRNA is lipid formulated in a formulation selected from Table A.
 116. The method of any one of claims 107-115, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation.
 117. The method of any one of claims 107-116, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously.
 118. The method of any one of claims 107-117, wherein the dsRNA is conjugated to GalNac.
 119. The method of any one of claims 107-118, wherein the dsRNA is conjugated to GalNac and administered subcutaneously.
 120. The method of any one of claims 107-119, wherein the dsRNA is administered subcutaneously.
 121. A method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of the dsRNA of any of claims 1 through
 29. 122. The method of claim 121, wherein the dsRNA is lipid formulated.
 123. The method of any one of claims 121-122, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation.
 124. The method of any one of claims 121-123, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously.
 125. The method of any one of claims 121-124, wherein the dsRNA is administered intravenously.
 126. The method of any one of claims 121-125, wherein the dsRNA is lipid formulated in a formulation selected from Table A.
 127. The method of any one of claims 121-126, wherein the dsRNA is conjugated to GalNac.
 128. The method of any one of claims 121-127, wherein the dsRNA is conjugated to GalNac and administered subcutaneously.
 129. The method of any one of claims 121-128, wherein the dsRNA is administered subcutaneously.
 130. The method of any one of claims 121-129, wherein the subject is a primate or a rodent.
 131. The method of any one of claims 121-130, wherein the subject is a human.
 132. The method of any one of claims 121-131, wherein the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.
 133. The method of any one of claims 121-132, wherein the subject has fatigue, shortness of breath, headache, dizziness, or pale skin.
 134. The method of any one of claims 121-133, wherein the subject has reduced iron levels compared to a subject without anemia.
 135. The method of any one of claims 121-134, wherein the subject has haemoglobin (Hb) levels <9 g/dL.
 136. The method of any one of claims 121-135, wherein the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA).
 137. The method of any one of claims 121-136, wherein the subject has reduced renal erythropoietin (EPO) synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD.
 138. The method of any one of claims 121-137, wherein the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD.
 139. The method of any one of claims 121-138, wherein the subject has a reticulocyte Hb of <28 pg.
 140. The method of any one of claims 121-139, wherein the subject has >10% hypochromic red blood cells (RBCs).
 141. The method of any one of claims 121-140, further comprising determining the complete blood count (CBC), serum iron concentration, Transferrin (Tf) saturation, or ferritin levels of the subject.
 142. The method of any one of claims 121-141, wherein administering results in an increase in iron levels in the subject.
 143. The method of any one of claims 121-142, wherein administering results in a 2-fold increase in iron levels in the subject.
 144. The method of any one of claims 121-143, wherein administering results in an increase in Tf saturation in the subject.
 145. The method of any one of claims 121-144, further comprising determining the iron level in the subject.
 146. The method of any one of claims 121-145, further comprising administering intravenous iron or ESAs to the subject.
 147. A method of inhibiting HFE2 expression in a cell, the method comprising: (a) introducing into the cell the dsRNA of any one of claims 36 through 61; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a HFE2 gene, thereby inhibiting expression of the HFE2 gene in the cell.
 148. The method of claim 147, wherein the HFE2 expression is inhibited by at least 30%.
 149. The method of any one of claims 147-148, wherein the HFE2 expression is inhibited by at least 80%.
 150. A method of treating a disorder associated with HFE2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of the dsRNA of any of claims 36 through
 61. 151. The method of claim 150, wherein the subject has anemia.
 152. The method of any one of claims 150-151, wherein the subject has refractory anemia.
 153. The method of any one of claims 150-152, wherein the subject has anemia of chronic disease (ACD).
 154. The method of any one of claims 150-153, wherein the subject has iron-restricted erythropoiesis.
 155. The method of any one of claims 150-154, wherein the subject is a human.
 156. The method of any one of claims 150-155, wherein the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
 157. The method of any one of claims 150-156, wherein the dsRNA is lipid formulated.
 158. The method of any one of claims 150-157, wherein the dsRNA is lipid formulated in a formulation selected from Table A.
 159. The method of any one of claims 150-158, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation.
 160. The method of any one of claims 150-159, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously.
 161. The method of any one of claims 150-160, wherein the dsRNA is conjugated to GalNac.
 162. The method of any one of claims 150-161, wherein the dsRNA is conjugated to GalNac and administered subcutaneously.
 163. The method of any one of claims 150-162, wherein the dsRNA is administered subcutaneously.
 164. A method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of the dsRNA of any of claims 36 through
 61. 165. The method of claim 164, wherein the dsRNA is lipid formulated.
 166. The method of any one of claims 164-165, wherein the dsRNA is lipid formulated in a formulation selected from Table A.
 167. The method of any one of claims 164-166, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation.
 168. The method of any one of claims 164-167, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously.
 169. The method of any one of claims 164-168, wherein the dsRNA is conjugated to GalNac.
 170. The method of any one of claims 164-169, wherein the dsRNA is conjugated to GalNac and administered subcutaneously.
 171. The method of any one of claims 164-170, wherein the dsRNA is administered subcutaneously.
 172. The method of any one of claims 164-171, wherein the subject is a primate or a rodent.
 173. The method of any one of claims 164-172, wherein the subject is a human.
 174. The method of any one of claims 164-173, wherein the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.
 175. The method of any one of claims 164-174, wherein the subject has fatigue, shortness of breath, headache, dizziness, or pale skin.
 176. The method of any one of claims 164-175, wherein the subject has reduced iron levels compared to a subject without anemia.
 177. The method of any one of claims 164-176, wherein the subject has Hb levels <9 g/dL.
 178. The method of any one of claims 164-177, wherein the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA).
 179. The method of any one of claims 164-178, wherein the subject has reduced renal EPO synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD.
 180. The method of any one of claims 164-179, wherein the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD.
 181. The method of any one of claims 164-180, wherein the subject has a reticulocyte Hb of <28 pg.
 182. The method of any one of claims 164-181, wherein the subject has >10% hypochromic RBCs.
 183. The method of any one of claims 164-182, further comprising determining the CBC, serum iron concentration, Transferrin (Tf) saturation, or ferritin levels of the subject.
 184. The method of any one of claims 164-183, wherein administering results in an increase in iron levels in the subject.
 185. The method of any one of claims 164-184, wherein administering results in a 2-fold increase in iron levels in the subject.
 186. The method of any one of claims 164-185, wherein administering results in an increase in Tf saturation in the subject.
 187. The method of any one of claims 164-186, further comprising determining the iron level in the subject.
 188. The method of any one of claims 164-187, further comprising administering intravenous iron or ESAs to the subject.
 189. A method of inhibiting TFR2 expression in a cell, the method comprising: (a) introducing into the cell the dsRNA of any one of claims 68 through 93; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TFR2 gene, thereby inhibiting expression of the TFR2 gene in the cell.
 190. The method of claim 189, wherein the TFR2 expression is inhibited by at least 30%.
 191. The method of any one of claims 189-190, wherein the TFR2 expression is inhibited by at least 80%.
 192. A method of treating a disorder associated with TFR2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of the dsRNA of any of claims 68 through
 93. 193. The method of claim 192, wherein the subject has anemia.
 194. The method of any one of claims 192-193, wherein the subject has refractory anemia.
 195. The method of any one of claims 192-194, wherein the subject has anemia of chronic disease (ACD).
 196. The method of any one of claims 192-195, wherein the subject has iron-restricted erythropoiesis.
 197. The method of any one of claims 192-196, wherein the subject is a human.
 198. The method of any one of claims 192-197, wherein the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
 199. The method of any one of claims 192-198, wherein the dsRNA is lipid formulated.
 200. The method of any one of claims 192-199, wherein the dsRNA is lipid formulated in a formulation selected from Table A.
 201. The method of any one of claims 192-200, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation.
 202. The method of any one of claims 192-201, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously.
 203. The method of any one of claims 192-202, wherein the dsRNA is conjugated to GalNac.
 204. The method of any one of claims 192-203, wherein the dsRNA is conjugated to GalNac and administered subcutaneously.
 205. The method of any one of claims 192-204, wherein the dsRNA is administered subcutaneously.
 206. A method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of the dsRNA of any of claims 68 through
 93. 207. The method of claim 206, wherein the dsRNA is lipid formulated.
 208. The method of any one of claims 206-207, wherein the dsRNA is lipid formulated in a formulation selected from Table A.
 209. The method of any one of claims 206-208, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation.
 210. The method of any one of claims 206-209, wherein the dsRNA is lipid formulated in a nucleic acid lipid particle formulation and administered intravenously.
 211. The method of any one of claims 206-210, wherein the dsRNA is conjugated to GalNac.
 212. The method of any one of claims 206-211, wherein the dsRNA is conjugated to GalNac and administered subcutaneously.
 213. The method of any one of claims 206-212, wherein the dsRNA is administered subcutaneously.
 214. The method of any one of claims 206-213, wherein the subject is a primate or a rodent.
 215. The method of any one of claims 206-214, wherein the subject is a human.
 216. The method of any one of claims 206-215, wherein the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.
 217. The method of any one of claims 206-216, wherein the subject has fatigue, shortness of breath, headache, dizziness, or pale skin.
 218. The method of any one of claims 206-217, wherein the subject has reduced iron levels compared to a subject without anemia.
 219. The method of any one of claims 206-218, wherein the subject has Hb levels <9 g/dL.
 220. The method of any one of claims 206-219, wherein the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA).
 221. The method of any one of claims 206-220, wherein the subject has reduced renal EPO synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD.
 222. The method of any one of claims 206-221, wherein the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD.
 223. The method of any one of claims 206-222, wherein the subject has a reticulocyte Hb of <28 pg.
 224. The method of any one of claims 206-223, wherein the subject has >10% hypochromic RBCs.
 225. The method of any one of claims 206-224, further comprising determining the CBC, serum iron concentration, Transferrin (Tf) saturation, or ferritin levels of the subject.
 226. The method of any one of claims 206-225, wherein administering results in an increase in iron levels in the subject.
 227. The method of any one of claims 206-226, wherein administering results in a 2-fold increase in iron levels in the subject.
 228. The method of any one of claims 206-227, wherein administering results in an increase in Tf saturation in the subject.
 229. The method of any one of claims 206-228, further comprising determining the iron level in the subject.
 230. The method of any one of claims 206-229, further comprising administering intravenous iron or ESAs to the subject.
 231. A method of inhibiting HAMP, HFE2, and/or TFR2 expression in a cell, the method comprising: (a) introducing into the cell a plurality of dsRNAs selected from the dsRNAs of any one of claims 1 through 29, 36 through 61, and 68 through 93; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a HAMP, HFE2, and/or TFR2 gene, thereby inhibiting expression of the HAMP, HFE2, and/or TFR2 gene in the cell.
 232. The method of claim 231, wherein the plurality of dsRNAs are introduced simultaneously.
 233. The method of any one of claims 231-232, wherein the plurality of dsRNAs are introduced concurrently.
 234. The method of any one of claims 231-233, wherein the plurality of dsRNAs are introduced individually.
 235. The method of any one of claims 231-234, wherein the plurality of dsRNAs are introduced together.
 236. The method of any one of claims 231-235, wherein the expression is inhibited by at least 30%.
 237. The method of any one of claims 231-236, wherein the expression is inhibited by at least 80%.
 238. A method of treating a disorder associated with HAMP, HFE2, and/or TFR2 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of a plurality of dsRNAs selected from the dsRNAs of any one of claims 1 through 29, 36 through 61, and 68 through
 93. 239. The method of claim 238, wherein the plurality of dsRNAs are administered to the subject simultaneously.
 240. The method of any one of claims 238-239, wherein the plurality of dsRNAs are administered to the subject concurrently.
 241. The method of any one of claims 238-240, wherein the plurality of dsRNAs are administered to the subject individually.
 242. The method of any one of claims 238-241, wherein the plurality of dsRNAs are administered to the subject together.
 243. The method of any one of claims 238-242, wherein the subject has anemia.
 244. The method of any one of claims 238-243, wherein the subject has refractory anemia.
 245. The method of any one of claims 238-244, wherein the subject has anemia of chronic disease (ACD).
 246. The method of any one of claims 238-245, wherein the subject has iron-restricted erythropoiesis.
 247. The method of any one of claims 238-246, wherein the subject is a human.
 248. The method of any one of claims 238-247, wherein the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
 249. A method for treating anemia in a subject in need thereof comprising administering to the subject an effective amount of a plurality of dsRNAs selected from the dsRNAs of any one of claims 1 through 29, 36 through 61, and 68 through
 93. 250. The method of claim 249, wherein the plurality of dsRNAs are administered to the subject simultaneously.
 251. The method of any one of claims 249-250, wherein the plurality of dsRNAs are administered to the subject concurrently.
 252. The method of any one of claims 249-251, wherein the plurality of dsRNAs are administered to the subject individually.
 253. The method of any one of claims 249-252, wherein the plurality of dsRNAs are administered to the subject together.
 254. The method of any one of claims 249-253, wherein the dsRNA is lipid formulated.
 255. The method of any one of claims 249-254, wherein the dsRNA is lipid formulated in a formulation selected from Table A.
 256. The method of any one of claims 249-255, wherein the subject is a primate or a rodent.
 257. The method of any one of claims 249-256, wherein the subject is a human.
 258. The method of any one of claims 249-257, wherein the effective amount is a concentration of 0.01-5.0 mg/kg bodyweight of the subject.
 259. The method of any one of claims 249-258, wherein the subject has fatigue, shortness of breath, headache, dizziness, or pale skin.
 260. The method of any one of claims 249-259, wherein the subject has reduced iron levels compared to a subject without anemia.
 261. The method of any one of claims 249-260, wherein the subject has Hb levels <9 g/dL.
 262. The method of any one of claims 249-261, wherein the subject has chronic kidney disease (CKD), cancer, chronic inflammatory disease, rheumatoid arthritis (RA), or iron-resistant iron-deficient amemia (IRIDA).
 263. The method of any one of claims 249-262, wherein the subject has reduced renal EPO synthesis compared to a subject without CKD, a dietary deficiency, blood loss, or elevated hepcidin levels compared to a subject without CKD.
 264. The method of any one of claims 249-263, wherein the subject has decreased renal excretion of hepcidin compared to a subject without CKD or low grade inflammation characterized by increased interleukin-6 (IL-6) levels compared to a subject without CKD.
 265. The method of any one of claims 249-264, wherein the subject has a reticulocyte Hb of <28 pg.
 266. The method of any one of claims 249-265, wherein the subject has >10% hypochromic RBCs.
 267. The method of any one of claims 249-266, further comprising determining the CBC, serum iron concentration, Transferrin (Tf) saturation, or ferritin levels of the subject.
 268. The method of any one of claims 249-267, wherein administering results in an increase in iron levels in the subject.
 269. The method of any one of claims 249-268, wherein administering results in a 2-fold increase in iron levels in the subject.
 270. The method of any one of claims 249-269, wherein administering results in an increase in Tf saturation in the subject.
 271. The method of any one of claims 249-270, further comprising determining the iron level in the subject.
 272. The method of any one of claims 249-271, further comprising administering intravenous iron or ESAs to the subject. 